Contributions of the Dachsous intracellular domain to Dachsous-Fat signaling.

The protocadherins Fat and Dachsous regulate organ growth, shape, patterning, and planar cell polarity. Although Dachsous and Fat have been described as ligand and receptor, respectively, in a signal transduction pathway, there is also evidence for bidirectional signaling. Here we assess signaling downstream of Dachsous through analysis of its intracellular domain. Genomic deletions of conserved sequences within dachsous identified regions of the intracellular domain required for normal development. Deletion of the A motif increased Dachsous protein levels and decreased wing size. Deletion of the D motif decreased Dachsous levels at cell membranes, increased wing size, and disrupted wing, leg and hindgut patterning and planar cell polarity. Co-immunoprecipitation experiments established that the D motif is necessary and sufficient for association of Dachsous with four key partners: Lowfat, Dachs, Spiny-legs, and MyoID. Subdivision of the D motif identified distinct regions that are preferentially responsible for association with Lft versus Dachs. Our results identify motifs that are essential for Dachsous function and are consistent with the hypothesis that the key function of Dachsous is regulation of Fat.

Competition between myosin II and ßH-spectrin regulates cytoskeletal tension.

Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two α-spectrins and two ß-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. We have investigated the role and regulation of the Drosophila ß-heavy spectrin (ßH-spectrin, encoded by the karst gene) in wing imaginal discs. Our results establish that ßH-spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While we find that α-spectrin also regulates Hippo signaling through Jub, unexpectedly, we find that ßH-spectrin localizes and functions independently of α-spectrin. Instead, ßH-spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which ßH-spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of ßH-spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how ßH-spectrin participates in ratcheting mechanisms associated with cell shape change.

AJUBA and WTIP can compete with LIMD1 for junctional localization and LATS regulation.

Each of the three mammalian Ajuba family proteins, AJUBA, LIMD1 and WTIP, exhibit tension-dependent localization to adherens junctions, and can associate with Lats kinases. However, only LIMD1 has been directly demonstrated to directly regulate Lats activity in vivo. To assess the relationship of LIMD1 to AJUBA and WTIP, and the potential contributions of AJUBA and WTIP to Lats regulation, we examined the consequences of over-expressing AJUBA and WTIP in MCF10A cells. Over-expression of either AJUBA or WTIP reduced junctional localization of LIMD1, implying that these proteins can compete for binding to adherens junctions. This over-expression also reduced junctional localization of LATS1, implying that AJUBA or WTIP are unable to efficiently recruit Lats kinases to adherens junctions. This over-expression was also associated with increased YAP1 phosphorylation and decreased YAP1 nuclear localization, consistent with increased Lats kinase activity. These observations indicate that AJUBA and WTIP compete with LIMD1 for association with adherens junctions but have activities distinct from LIMD1 in Hippo pathway regulation. They further suggest that the ability of Ajuba family proteins to associate with Lats kinases in solution is not sufficient to enable regulation in vivo, and that tumor suppressor activities of AJUBA and WTIP could stem in part from competition with LIMD1 for regulation of Lats kinases at cell junctions.

Near-infrared nuclear markers for Drosophila imaging.

Nuclear markers for live imaging are useful for counting and tracking cells, visualizing cell division, and examining the regulation of proteins that are controlled via entry or exit from the nucleus. Near-infrared fluorescent proteins have advantages over shorter wavelength fluorescent proteins, including reduced phototoxicity, less light scattering, and enabling multicolor live imaging. We have constructed and tested transgenic Drosophila expressing Histone H2Av iRFP fusion proteins, and confirmed that they can be used to label nuclei in both fixed and live tissue at multiple stages of development.

Analysis of the Drosophila Ajuba LIM protein defines functions for distinct LIM domains.

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.

Investigating the effect of Eh and pH on binding forms of Co, Cu, and Pb in wetland sediments from Zambia.

This study investigated binding forms of cobalt (Co), copper (Cu), and lead (Pb) in 28 sediment samples from inlet to outlet of three Zambian wetlands receiving mining effluents. Use was made of a modified Tessier metal binding fractions procedure. Due to storage artefacts, the original aim of investigating the effects of redox potential (Eh) changes, starting from extremely low Eh, was suspended. Instead, use was made of the new, not often explored opportunity for replicate sample division into three categories of varying redox potential and pH. Additionally, in line with the original research aim, two sediments from each wetland were investigated for their response to increasing Eh. The results showed overall high trace metal contents, with a need for remedial actions for Co and Cu in the first, Cu in the second, and Pb in the third wetland. Rather independent of Eh and pH, Co was often found in the residual fraction (F5), as well as in the oxidizable (F4) and reducible (F3) fraction. Cu was generally dominant in F5 and F4 fractions, with low F3 prevalence, indicating a high organic matter affinity. Pb distribution among binding forms showed small variations within and across wetlands, F5, F4, and F3 fractions dominating. In the above observations, statistical analysis showed that, among the 28 sediment samples across wetlands, the influence of Eh and pH on binding forms were generally found to be not significant, being 'overruled' by other sedimentological factors. With increasing Eh, the decrease in the oxidizable (F4) fraction was smaller than expected in eight of 18 tests. The Risk Assessment Code (RAC) method, based on the exchangeable fraction (F1) plus carbonate fraction (F2), showed that some sediments turned from "unsafe" to "safe," and vice versa, with increasing Eh. The "total metals method" does not show bioavailability, whereas RAC does not use the metal contents. Thus, the two methods should be used together to improve the prediction of potential toxicity.

The wing imaginal disc.

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.

Distribution of the heavy metals Co, Cu, and Pb in sediments and Typha spp. And Phragmites mauritianus in three Zambian wetlands.

Zambia has been mining cobalt (Co), copper (Cu), and lead (Pb) for over a century, with discharges entering wetlands without investigations on the level of sediment pollution and how to solve it. This present study investigated: 1) the extent to which Co, Cu, and Pb that enter through mining wastewater were distributed in the sediment of three wetlands (Uchi, Mufulira, and Kabwe) in Zambia and 2) the accumulation and distribution of the heavy metals in two emergent wetland plants, Phragmites mauritianus, and Typha spp. in order to evaluate their potential for phytoremediation of metals. Samples from three sections (inlet, middle section and outlet) of each wetland were analyzed for the heavy metal contents. Sediment contents of Co and Cu were significantly higher in the Uchi wetland than in the other two, while Pb was significantly higher in the Kabwe wetland. Cu in all the wetlands were found to be at levels considered a threat to aquatic life, with Pb contents in Kabwe a risk to human health. Both P. mauritianus and Typha spp acted as excluder species for Co, Cu, and Pb, showing bioaccumulation factor (BAF) < 1 and Translocation factor (TF) < 1 for all wetlands. As neither species accumulated cellularly toxic concentrations of Co, Cu, and Pb, they could grow in the contaminated sediments. Currently, methods used to solve historic mining impacts in Zambian wetlands aim at improving water flow and reducing flooding without attending to the heavy metal contents of the sediments. From this study, P. mauritianus and Typha spp. provide the potential for phytostabilisation to settle and contain polluted sediments.

TRIP6 is required for tension at adherens junctions.

Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.

E2 and Gamma distributions in polygonal networks.

From solar supergranulation to salt flat in Bolivia, from veins on leaves to cells on Drosophila wing discs, polygon-based networks exhibit great complexities, yet similarities and consistent patterns emerge. Based on analysis of 99 polygonal tessellations of a wide variety of physical origins, this work demonstrates the ubiquity of an exponential distribution in the squared norm of the deformation tensor, E2, which directly leads to the ubiquitous presence of Gamma distributions in polygon aspect ratio as recently demonstrated by Atia et al. [Nat. Phys. 14, 613 (2018)]. In turn an analytical approach is developed to illustrate its origin. E2 relates to most energy forms, and its Boltzmann-like feature allows the definition of a pseudo-temperature that promises utility in a thermodynamic ensemble framework.

Distribution patterns of epiphytic reed-associated macroinvertebrate communities across European shallow lakes.

So far, research on plant-associated macroinvertebrates, even if conducted on a large number of water bodies, has mostly focused on a relatively small area, permitting limited conclusions to be drawn regarding potentially broader geographic effects, including climate. Some recent studies have shown that the composition of epiphytic communities may differ considerably among climatic zones. To assess this phenomenon, we studied macroinvertebrates associated with the common reed Phragmites australis (Cav.) Trin. ex Steud in 46 shallow lakes using a common protocol. The lakes, located in nine countries, covered almost the entire European latitudinal range (from <48°N to 61°N) and captured much of the variability in lake size and nutrient content in the region. A Poisson Generalized Linear Mixed Model (GLMM) showed the number of macroinvertebrate epiphytic taxa to be negatively associated with water conductivity and positively associated with medium ice cover duration (approximately 1 month). A Gamma GLMM showed a positive effect of chlorophyll a on the density of macroinvertebrates, and a significantly greater density in lakes located at the lowest and highest latitudes. Individual taxa responded differently to lake environmental conditions across climate zones. Chironomidae dominated in all climate zones, but their contribution to total density decreased with increasing latitude, with progressively greater proportions of Naidinae, Asellidae, Ephemeroptera and Trichoptera. Our study demonstrates that epiphytic macroinvertebrate fauna, even when analyzed at low taxonomic resolution, exhibits clear differences in diversity, relative abundance of individual taxa and total density, shaped both by geographic and anthropogenic variables. The results were discussed in the context of climate change. To our best knowledge this is the first study to examine epiphytic fauna carried out on a European scale.

Integration of Hippo-YAP Signaling with Metabolism.

The Hippo-Yes-associated protein (YAP) signaling network plays a central role as an integrator of signals that control cellular proliferation and differentiation. The past several years have provided an increasing appreciation and understanding of the diverse mechanisms through which metabolites and metabolic signals influence Hippo-YAP signaling, and how Hippo-YAP signaling, in turn, controls genes that direct cellular and organismal metabolism. These connections enable Hippo-YAP signaling to coordinate organ growth and homeostasis with nutrition and metabolism. In this review, we discuss the current understanding of some of the many interconnections between Hippo-YAP signaling and metabolism and how they are affected in disease conditions.

Capacity challenges in water quality monitoring: understanding the role of human development.

Monitoring the qualitative status of freshwaters is an important goal of the international community, as stated in the Sustainable Development Goal (SDGs) indicator 6.3.2 on good ambient water quality. Monitoring data are, however, lacking in many countries, allegedly because of capacity challenges of less-developed countries. So far, however, the relationship between human development and capacity challenges for water quality monitoring have not been analysed systematically. This hinders the implementation of fine-tuned capacity development programmes for water quality monitoring. Against this background, this study takes a global perspective in analysing the link between human development and the capacity challenges countries face in their national water quality monitoring programmes. The analysis is based on the latest data on the human development index and an international online survey amongst experts from science and practice. Results provide evidence of a negative relationship between human development and the capacity challenges to meet SDG 6.3.2 monitoring requirements. This negative relationship increases along the course of the monitoring process, from defining the enabling environment, choosing parameters for the collection of field data, to the analytics and analysis of five commonly used parameters (DO, EC, pH, TP and TN). Our assessment can be used to help practitioners improve technical capacity development activities and to identify and target investment in capacity development for monitoring.

Dchs1-Fat4 regulation of osteogenic differentiation in mouse.

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of Yap-Runx2 and Taz-Runx2 complexes is altered in Dchs1/Fat4 mutant osteoblasts. In conclusion, these data identify Dchs1-Fat4 as a signalling pathway in osteoblast differentiation, reveal its crucial role within the early Runx2 progenitors, and identify distinct requirements for Yap and Taz during osteoblast differentiation.

Organization and function of tension-dependent complexes at adherens junctions.

Adherens junctions provide attachments between neighboring epithelial cells and a physical link to the cytoskeleton, which enables them to sense and transmit forces and to initiate biomechanical signaling. Examination of the Ajuba LIM protein Jub in Drosophila embryos revealed that it is recruited to adherens junctions in tissues experiencing high levels of myosin activity, and that the pattern of Jub recruitment varies depending upon how tension is organized. In cells with high junctional myosin, Jub is recruited to puncta near intercellular vertices, which are distinct from Ena-containing puncta, but can overlap Vinc-containing puncta. We identify roles for Jub in modulating tension and cellular organization, which are shared with the cytohesin Step, and the cytohesin adapter Sstn, and show that Jub and Sstn together recruit Step to adherens junctions under tension. Our observations establish Jub as a reporter of tension experienced at adherens junctions, and identify distinct types of tension-dependent and tension-independent junctional complexes. They also identify a role for Jub in mediating a feedback loop that modulates the distribution of tension and cellular organization in epithelia.

Investigating Co, Cu, and Pb retention and remobilization after drying and rewetting treatments in greenhouse laboratory-scale constructed treatments with and without Typha angustifolia, and connected phytoremediation potential.

There is critical concern over heavy metals because they are biotoxins. The best management option is elimination or at least minimization of effluence into the environment, but in several regions, mining wastewater or acid mine drainage (AMD) effluence into natural wetlands has continued. The ability of wetlands to attenuate heavy metals in mining wastewater and AMD has led to natural wetlands being used as recipients of these effluents in many parts of the world. Ten greenhouse-based laboratory-scale constructed wetlands (GLCW) were set up at IHE-Delft Institute for Water Education to understand the mechanisms and fate of heavy metals in three Zambian wetlands in attenuation of Co, Cu, and Pb. These were operated as Free Water Surface Constructed Wetlands (FWS-CWs). The principal investigations compared how vegetated and unvegetated microcosm artificial wetlands retained controlled additions of heavy metals and the effect of drying and rewetting on that. The potential for phytoremediation using Typha angustifolia was also investigated. Typha angustifolia was planted in three vegetated and compared with one unvegetated treatment. Treatments A, B, and, the investigated, Treatment D received synthetic wastewater containing Co, Cu, and Pb, while a control, Treatment C, received tap water. Water samples were taken throughout the experiment, and sediment samples collected after the first flushing and before drying. Samples of T. angustifolia were taken before drying the wetlands. Analyses for Co, Cu, and Pb were made in the water and sediment, and in roots, stems and leaves of plant samples. The unvegetated Dutch sediments GLCWs removed more Co from wastewater (52%) than the vegetated Dutch and Zambian sediments GLCWs (13% and -4%, respectively). There was a similar removal of Cu among the GLCWs receiving wastewater (81%-87%). The removal of Pb was significantly higher in the vegetated Dutch sediment GLCWs than the unvegetated Dutch sediments GLCWs, (89% and 72%, respectively). It was concluded that a hectare of the vegetated Zambian sediments with similar design parameters of 50 mg/m2.day for Co, Cu, and Pb used in the experiment would on average retain 83 g/day of Co, and 417 g/day of both Cu and Pb. After drying, Co, Cu, and Pb washed out on the first day of rewetting. The washout after that took only a few days. How long the metals washed out of the GLCWs was in order Co > Cu > Pb. T. angustifolia could neither be classified as an accumulator nor an excluder species because the concentrations of Co, Cu, and Pb in the sediments and T. angustifolia were below phytotoxic levels mainly due to a short running period of the experiment.

Oriented Cell Divisions Are Not Required for Drosophila Wing Shape.

Formation of correctly shaped organs is vital for normal function. The Drosophila wing has an elongated shape, which has been attributed in part to a preferential orientation of mitotic spindles along the proximal-distal axis [1, 2]. Orientation of mitotic spindles is believed to be a fundamental morphogenetic mechanism in multicellular organisms [3-6]. A contribution of spindle orientation to wing shape was inferred from observations that mutation of Dachsous-Fat pathway genes results in both rounder wings and loss of the normal proximal-distal bias in spindle orientation [1, 2, 7]. To directly evaluate the potential contribution of spindle orientation to wing morphogenesis, we assessed the consequences of loss of the Drosophila NuMA homolog Mud, which interacts with the dynein complex and has a conserved role in spindle orientation [8, 9]. Loss of Mud randomizes spindle orientation but does not alter wing shape. Analysis of growth and cell dynamics in developing discs and in ex vivo culture suggests that the absence of oriented cell divisions is compensated for by an increased contribution of cell rearrangements to wing shape. Our results indicate that oriented cell divisions are not required for wing morphogenesis, nor are they required for the morphogenesis of other Drosophila appendages. Moreover, our results suggest that normal organ shape is not achieved through locally specifying and then summing up individual cell behaviors, like oriented cell division. Instead, wing shape might be specified through tissue-wide stresses that dictate an overall arrangement of cells without specifying the individual cell behaviors needed to achieve it.

Early girl is a novel component of the Fat signaling pathway.

The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. Here we identify the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. Our findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway.

Recruitment of Jub by α-catenin promotes Yki activity and Drosophila wing growth.

The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for the biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions. Here, we investigate the mechanism by which the Drosophila Ajuba family protein Jub is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. We identify α-catenin as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. We also show that increased Jub recruitment to α-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. Our observations establish α-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to α-catenin as a key contributor to biomechanical regulation of Hippo signaling.

Localization of Hippo Signaling Components in Drosophila by Fluorescence and Immunofluorescence.

Visualization of in vivo protein levels and localization is essential to analysis and elucidation of Hippo signaling mechanisms and its roles in diverse tissues. This is best done by imaging proteins using fluorescent labels. Fluorescent labeling of a protein can be achieved by direct conjugation to an intrinsically fluorescent protein, like GFP, or by use of antibodies conjugated to fluorescent dyes. Immunofluorescence imaging in Drosophila typically begins with dissection and fixation of a sample tissue, followed by a series of washes and incubations with primary antibodies, directed against proteins of interest, and dye-labeled secondary antibodies, directed against the primary antibodies. This may be followed by fluorescent dyes that label cellular components, such as DNA-labeling dyes to mark nuclei. After staining and washing is completed, samples are placed in a mounting media, transferred to a microscope slide, and imaged on a confocal microscope.