Transcriptome analysis reveals temporally regulated genetic networks during Drosophila border cell collective migration.

BACKGROUND: Collective cell migration underlies many essential processes, including sculpting organs during embryogenesis, wound healing in the adult, and metastasis of cancer cells. At mid-oogenesis, Drosophila border cells undergo collective migration. Border cells round up into a small group at the pre-migration stage, detach from the epithelium and undergo a dynamic and highly regulated migration at the mid-migration stage, and stop at the oocyte, their final destination, at the post-migration stage. While specific genes that promote cell signaling, polarization of the cluster, formation of protrusions, and cell-cell adhesion are known to regulate border cell migration, there may be additional genes that promote these distinct active phases of border cell migration. Therefore, we sought to identify genes whose expression patterns changed during border cell migration. RESULTS: We performed RNA-sequencing on border cells isolated at pre-, mid-, and post-migration stages. We report that 1,729 transcripts, in nine co-expression gene clusters, are temporally and differentially expressed across the three migration stages. Gene ontology analyses and constructed protein-protein interaction networks identified genes expected to function in collective migration, such as regulators of the cytoskeleton, adhesion, and tissue morphogenesis, but also uncovered a notable enrichment of genes involved in immune signaling, ribosome biogenesis, and stress responses. Finally, we validated the in vivo expression and function of a subset of identified genes in border cells. CONCLUSIONS: Overall, our results identified differentially and temporally expressed genetic networks that may facilitate the efficient development and migration of border cells. The genes identified here represent a wealth of new candidates to investigate the molecular nature of dynamic collective cell migrations in developing tissues.

Singed and vinculin play redundant roles in cell migration by regulating F-actin.

INTRODUCTION: Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin-1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. RESULT: In this work, we have screened many actin-binding proteins in search of functional redundancy with Singed for border cell migration. We have found that Vinculin works with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F-actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F-actin and changes in protrusion characteristics in border cells. We have also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. CONCLUSIONS: We may conclude that singed and vinculin work together to control F-actin and these interactions are consistent across multiple platforms.

A biochemical network controlling basal myosin oscillation.

The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. Here, we show that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, we uncover an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation.

Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue.

Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6-10 cells, traversing a network of large germ line-derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues.

Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration.

BACKGROUND: Localized actomyosin contraction couples with actin polymerization and cell-matrix adhesion to regulate cell protrusions and retract trailing edges of migrating cells. Although many cells migrate in collective groups during tissue morphogenesis, mechanisms that coordinate actomyosin dynamics in collective cell migration are poorly understood. Migration of Drosophila border cells, a genetically tractable model for collective cell migration, requires nonmuscle myosin-II (Myo-II). How Myo-II specifically controls border cell migration and how Myo-II is itself regulated is largely unknown. RESULTS: We show that Myo-II regulates two essential features of border cell migration: (1) initial detachment of the border cell cluster from the follicular epithelium and (2) the dynamics of cellular protrusions. We further demonstrate that the cell polarity protein Par-1 (MARK), a serine-threonine kinase, regulates the localization and activation of Myo-II in border cells. Par-1 binds to myosin phosphatase and phosphorylates it at a known inactivating site. Par-1 thus promotes phosphorylated myosin regulatory light chain, thereby increasing Myo-II activity. Furthermore, Par-1 localizes to and increases active Myo-II at the cluster rear to promote detachment; in the absence of Par-1, spatially distinct active Myo-II is lost. CONCLUSIONS: We identify a critical new role for Par-1 kinase: spatiotemporal regulation of Myo-II activity within the border cell cluster through localized inhibition of myosin phosphatase. Polarity proteins such as Par-1, which intrinsically localize, can thus directly modulate the actomyosin dynamics required for border cell detachment and migration. Such a link between polarity proteins and cytoskeletal dynamics may also occur in other collective cell migrations.

Allergenicity assessment of Allium sativum leaf agglutinin, a potential candidate protein for developing sap sucking insect resistant food crops.

BACKGROUND: Mannose-binding Allium sativum leaf agglutinin (ASAL) is highly antinutritional and toxic to various phloem-feeding hemipteran insects. ASAL has been expressed in a number of agriculturally important crops to develop resistance against those insects. Awareness of the safety aspect of ASAL is absolutely essential for developing ASAL transgenic plants. METHODOLOGY/PRINCIPAL FINDINGS: Following the guidelines framed by the Food and Agriculture Organization/World Health Organization, the source of the gene, its sequence homology with potent allergens, clinical tests on mammalian systems, and the pepsin resistance and thermostability of the protein were considered to address the issue. No significant homology to the ASAL sequence was detected when compared to known allergenic proteins. The ELISA of blood sera collected from known allergy patients also failed to show significant evidence of cross-reactivity. In vitro and in vivo assays both indicated the digestibility of ASAL in the presence of pepsin in a minimum time period. CONCLUSIONS/SIGNIFICANCE: With these experiments, we concluded that ASAL does not possess any apparent features of an allergen. This is the first report regarding the monitoring of the allergenicity of any mannose-binding monocot lectin having insecticidal efficacy against hemipteran insects.

Src inhibitor dasatinib inhibits growth of breast cancer cells by modulating EGFR signaling.

EGF-receptor family members (EGFRs) as well as c-Src are over expressed in approximately 70% of breast cancer, and in most of the tumors c-Src is co-over expressed with at least one of the EGFRs, suggesting that they may interact functionally and play a role in the development and progression of the malignancy. We hypothesize that a small molecule inhibitor of c-Src dasatinib (BMS-354825; Bristol Myers Squibb), exerts its effects on breast cancer cells by modulating EGFR signaling. Indeed, we found that dasatinib causes inhibition of breast cancer cells overexpressing EGFR, HER-2 and HER-3 (MDA-MB-468, SKBR3, MDA-MB-453, and MDA-MB-231) in a dose and time-dependent manner. Dasatinib also stimulated apoptosis in MDA-MB-468 cells, which could be attributed to activation of both caspase-9 and -8 and arrest of the cell cycle at G0/G1 cycle. Furthermore, dasatinib markedly inhibited colony formation, cell invasion, migration and angiogenesis, accompanied by decreased phosphorylation of EGFR and c-Src and their downstream effector molecules Akt and Erks. Our data suggest that dasatinib mediates its action in part through EGFR signaling and could be a potential therapeutic agent for breast cancer.

Transactivator of transcription-tagged cell cycle and apoptosis regulatory protein-1 peptides suppress the growth of human breast cancer cells in vitro and in vivo.

Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)-tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1-198, 197-454, and 896-1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1-198(Y192/F)) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1-198(Y192/F)), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1-198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1-198(Y192/F)), inhibits growth of human breast cancer cell-derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing.

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests.

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be approximately 12.1%+/-0.351 (mean +/- SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P < 0.01), 32% (P < 0.05) and 40.5, 29.5% (P < 0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

Insecticidal activity of Arum maculatum tuber lectin and its binding to the glycosylated insect gut receptors.

Mannose binding approximately 50 kDa homotetrameric lectin, purified from edible Arum maculatum tuber, was analyzed through SDS-PAGE and studied for its agglutination property using rabbit erythrocytes. Cross reactivity of the purified lectin was verified through western blot using Colocasia esculantum(Family, Araceae) tuber lectin antibody. The insecticidal activity of Arum maculatum tuber lectin (ATL) was tested against two economically important sucking pests, Lipaphis erysimi and Aphis craccivora, in an artificial diet. The LC(50) values for L. erysimi and A. craccivora were determined to be 21 microg/mL and 16 microg/mL, respectively. Addition of alpha-d-mannose in ATL-supplemented diet reduced the aphid mortality. Two major receptor proteins of ATL (approximately 40 kDa and approximately 35 kDa) were detected from the brush border membrane vesicle (BBMV) protein of L. erysimi and A. craccivora guts, respectively, using ligand-binding assay. Alpha-d-Mannose was found to be a deterrent to such binding of ATL to the BBMV receptors.

The efficacy of a novel insecticidal protein, Allium sativum leaf lectin (ASAL), against homopteran insects monitored in transgenic tobacco.

The homopteran group of polyphagous sucking insect pests causes severe damage to many economically important plants including tobacco. Allium sativum leaf lectin (ASAL), a mannose-binding 25-kDa homodimeric protein, has recently been found to be antagonistic to various sucking insects in the homopteran group through artificial diet bioassay experiments. The present study describes, for the first time, the expression of the ASAL coding sequence under the control of the cauliflower mosaic virus (CaMV) 35S promoter in tobacco by Agrobacterium-mediated transformation technology. Molecular analyses demonstrated the integration of the chimeric ASAL gene in tobacco and its inheritance in the progeny plants. Western blot analysis followed by enzyme-linked immunosorbent assay (ELISA) determined the level of ASAL expression in different lines to be in the range of approximately 0.68%-2% of total soluble plant protein. An in planta bioassay conducted with Myzus persicae, peach potato aphid (a devastating pest of tobacco and many other important plants), revealed that the percentage of insect survival decreased significantly to 16%-20% in T0 plants and T1 progeny, whilst approximately 75% of insects survived on untransformed tobacco plants after 144 h of incubation. Ligand analyses of insect brush border membrane vesicle receptors and expressed ASAL in transgenic tobacco showed that the expressed ASAL binds to the aphid gut receptor in the same manner as native ASAL, pointing to the fact that ASAL maintains the biochemical characteristics even in the transgenic situation. These findings in a model plant open up the possibility of expressing the novel ASAL gene in a wide range of crop plants susceptible to various sap-sucking insects.

Identification of receptors responsible for binding of the mannose specific lectin to the gut epithelial membrane of the target insects.

The sap-sucking homopteran insects, commonly known as aphids and leafhoppers are responsible for a huge amount of lost productivity of mustard, chickpea, cabbage, rice and many other important crops. Due to their unique feeding habits and ability to build up a huge population in a very short time, they are very difficult to control. The objective of the ongoing program is to develop insect-resistant crop species through genetic engineering techniques to combat the yield losses, which necessitates the identification of appropriate control elements. In this direction, mannose-binding 25 kDa lectins have been purified from leaves of garlic, Diffenbachia sequina and tubers of Colocasia esculanta. The purified lectins have been analyzed in SDS-PAGE. The effectiveness of these lectins against chickpea aphids, mustard aphids and green leaf hoppers of rice have been tested. The LC(50) value of each lectin against different insects had been monitored [1,2]. Through immunolocalization analysis, the binding of the lectin had been demonstrated at the epithelial membrane of the midgut of the lectin-treated insects [1]. Receptor proteins of brush border membrane vesicle (BBMV) of the target insects, responsible for binding of the lectin to the midgut of the epithelial layer have been purified and analyzed through ligand assay. Biochemical studies have been undertaken to investigate the lectin-receptor interaction at molecular level.

The Interactions of Allium sativum leaf agglutinin with a chaperonin group of unique receptor protein isolated from a bacterial endosymbiont of the mustard aphid.

The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops. This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop. The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L. erysimi. The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut. Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry. Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein. To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program. The interaction was confirmed through docking of the two molecules forming a complex. A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule. This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission. Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission.

Efficiency of mannose-binding plant lectins in controlling a homopteran insect, the red cotton bug.

Yield losses of different crops due to the attack of various classes of insects are a worldwide problem. Sucking type homopteran pests causing damage to many crop species are not controlled by commonly known insecticidal proteins, namely, Bacillus thuringiensis delta-endotoxin (Bt). This study describes the purification of mannose-binding lectins from three different monocotyledonous plants (Allium sativum, Colocasia esculenta, and Diffenbachia sequina) and their effects on a homopteran insect, the red cotton bug. All of them had a detrimental effect on the growth and development of the insect, where A. sativum bulb lectin showed the highest mortality of all, in particular. The same bulb lectin not only affected the growth and fecundity of the insect but also imparted drastic changes in the color, weight, and size, even on the second generation of the insects which have been reared on artificial diet supplemented with a sublethal dose of the lectin. Thus, this finding opens up a possibility of using this lectin as an important component in crop management.