Rap1 maintains adhesion between cells to affect Egfr signaling and planar cell polarity in Drosophila

The small GTPase Rap1 affects cell adhesion and cell motility in numerous developmental contexts. Loss of Rap1 in the Drosophila wing epithelium disrupts adherens junction localization, causing mutant cells to disperse, and dramatically alters epithelial cell shape. While the adhesive consequences of Rap1 inactivation have been well described in this system, the effects on cell signaling, cell fate specification, and tissue differentiation are not known. Here we demonstrate that Egfr-dependent cell types are lost from Rap1 mutant tissue as an indirect consequence of DE-cadherin mislocalization. Cells lacking Rap1 in the developing wing and eye are capable of responding to an Egfr signal, indicating that Rap1 is not required for Egfr/Ras/MAPK signal transduction. Instead, Rap1 regulates adhesive contacts necessary for maintenance of Egfr signaling between cells, and differentiation of wing veins and photoreceptors. Rap1 is also necessary for planar cell polarity in these tissues. Wing hair alignment and ommatidial rotation, functional readouts of planar cell polarity in the wing and eye respectively, are both affected in Rap1 mutant tissue. Finally, we show that Rap1 acts through the effector Canoe to regulate these developmental processes.     

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.     

Lineage fusion in Galápagos giant tortoises

Although many classic radiations on islands are thought to be the result of repeated lineage splitting, the role of past fusion is rarely known because during these events, purebreds are rapidly replaced by a swarm of admixed individuals. Here, we capture lineage fusion in action in a Galápagos giant tortoise species, Chelonoidis becki, from Wolf Volcano (Isabela Island). The long generation time of Galápagos tortoises and dense sampling (841 individuals) of genetic and demographic data were integral in detecting and characterizing this phenomenon. In C. becki, we identified two genetically distinct, morphologically cryptic lineages. Historical reconstructions show that they colonized Wolf Volcano from Santiago Island in two temporally separated events, the first estimated to have occurred ~199 000 years ago. Following arrival of the second wave of colonists, both lineages coexisted for approximately ~53 000 years. Within that time, they began fusing back together, as microsatellite data reveal widespread introgressive hybridization. Interestingly, greater mate selectivity seems to be exhibited by purebred females of one of the lineages. Forward‐in‐time simulations predict rapid extinction of the early arriving lineage. This study provides a rare example of reticulate evolution in action and underscores the power of population genetics for understanding the past, present and future consequences of evolutionary phenomena associated with lineage fusion.     

Measurement of the temperature of the resting rotor in analytical ultracentrifugation

Accurate measurements of rotor temperature are critical for the interpretation of hydrodynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor using iButton temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2 °C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.     

Revision of the amphiamerican Neotetraonchus Bravo-Hollis, 1968 (Monogenoidea: Dactylogyridae), with a description of N. vegrandis n. sp. from the gill lamellae of the blue sea catfish Ariopsis guatemalensis (Siluriformes: Ariidae) off the Pacific Coast of Mexico

Neotetraonchus Bravo-Hollis, 1968 is revised and reassigned to the Dactylogyridae Bychowsky, 1933 based on examinations of specimens representing four species from the gill lamellae of sea catfishes (Ariidae). The monotypic Neotetraonchidae Bravo-Hollis, 1968 is placed in synonymy with the Dactylogyridae. Neotetraonchus bychowskyi Bravo-Hollis, 1968 (type-species), is redescribed from the tete sea catfish Ariopsis seemanni (Günther) (type-host) in the eastern Pacific Ocean off Panama (new geographical record). Neotetraonchus vegrandis n. sp. is described from the blue sea catfish A. guatemalensis (Günther) off the Pacific Coast of Mexico. Neotetraonchus bravohollisae Paperna, 1977 is redescribed from the hardhead sea catfish A. felis (L.) in the Gulf of Mexico off the Yucatan Peninsula. Neotetraonchus felis (Hargis, 1955) Paperna, 1977 is redescribed from A. felis in the Gulf of Mexico off Mississippi and the Yucatan Peninsula (new geographical record). Morphological similarities between species of Neotetraonchus suggest the likely presence of geminate species pairs flanking the Isthmus of Panama.

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Signal Transduction in the Footsteps of Goethe and Schiller

The historical town of Weimar in Thuringia, the "green heart of Germany" was the sphere of Goethe and Schiller, the two most famous representatives of German literature's classic era. Not yet entirely as influential as those two cultural icons, the Signal Transduction Society (STS) has nevertheless in the last decade established within the walls of Weimar an annual interdisciplinary Meeting on "Signal Transduction – Receptors, Mediators and Genes", which is well recognized as a most attractive opportunity to exchange results and ideas in the field.The 12^th STS Meeting was held from October 28 to 31 and provided a state-of-the-art overview of various areas of signal transduction research in which progress is fast and discussion lively. This report is intended to share with the readers of CCS some highlights of the Meeting Workshops devoted to specific aspects of signal transduction.     

Rac1 Is Essential for Basement Membrane-Dependent Epiblast Survival

During murine peri-implantation development, the egg cylinder forms from a solid cell mass by the apoptotic removal of inner cells that do not contact the basement membrane (BM) and the selective survival of the epiblast epithelium, which does. The signaling pathways that mediate this fundamental biological process are largely unknown. Here we demonstrate that Rac1 ablation in embryonic stem cell-derived embryoid bodies (EBs) leads to massive apoptosis of epiblast cells in contact with the BM. Expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 additionally blocks the apoptosis of inner cells and cavitation, indicating that the spatially regulated activation of Rac1 is required for epithelial cyst formation. We further show that Rac1 is activated through integrin-mediated recruitment of the Crk-DOCK180 complex and mediates BM-dependent epiblast survival through activating the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Our results reveal a signaling cascade triggered by cell-BM interactions essential for epithelial morphogenesis.All epithelial sheets and tubes rest upon a basement membrane (BM), a thin mat of specialized extracellular matrix (ECM) consisting of laminins, type IV collagens, perlecan, and nidogens. The BM provides essential survival signals to protect epithelial cells from apoptosis, in addition to its role in cell adhesion, migration, proliferation, and polarity orientation. In the developing chick retina, removal of the retinal BM by collagenase digestion resulted in severe apoptosis of retinal neuroepithelial cells (17). In mice, targeted deletion of the genes for the BM component laminins or perlecan caused BM defects and various degrees of apoptosis of cells that attach to the BM (34, 41, 42). Also, mammary epithelial cells can survive for a long period of time when grown on a reconstituted basement membrane derived from Engelbreth-Holmof Swarm (EHS) tumor (Matrigel), but they die by apoptosis when grown on plastic, fibronectin, or type I collagen despite their firm attachment on these substrates (2, 11, 36). A similar response of keratinocytes to BM type IV collagen versus non-BM matrix proteins was observed in bioengineered human skin equivalents (40). These results suggest that the BM provides a unique microenvironment for the survival of associated epithelial cells.Embryoid body (EB) differentiation has been used to study epithelial morphogenesis and early embryogenesis. When cultured in suspension as small aggregates, mouse embryonic stem (ES) cells adhere strongly together and form spherical EBs. The outer cells of the EB differentiate to become endoderm cells, which secrete laminins, type IV collagen, perlecan, and other BM components that assemble into an underlying BM equivalent to the embryonic BM separating extraembryonic endoderm from the epiblast. Integrin α6β1 in the epiblast cells and integrin α5β1 in the endoderm cells redistribute from a pericellular location to a predominantly sub-basement membrane location (28). Following BM formation, the epiblast cells adjacent to the BM polarize to become a pseudostratified columnar epithelium (the epiblast epithelium), whereas the inner cells not in contact with the BM undergo apoptosis and are selectively removed by phagocytosis/autophagy, creating a proamniotic-like cavity. That the BM is essential for these sequential processes is evidenced by the observation that targeted deletion of the laminin γ1 gene in EBs blocks BM assembly, subsequent epiblast epithelialization, and then apoptosis-dependent cavitation (32, 42). These differentiation processes recapitulate peri-implantation development and provide a tractable in vitro model for the study of apoptosis and BM-dependent cell survival during epithelial morphogenesis.While BM-dependent cell survival is often coupled with apoptotic removal of centrally located cells not in contact with the BM during morphogenesis of epithelial cysts such as mammary glandular acini and embryonic mouse egg cylinders (7, 29), the molecular mechanisms underlying this fundamental process are poorly understood. Elegant studies on teratocarcinoma cell-derived EBs have suggested that formation of an epithelial cyst as they develop is the result of the interplay of two signals (7). One is a death signal from the endoderm that induces apoptosis of the centrally located cells to create a cavity; the other is a rescue signal mediated by contact with the BM and is required for the survival of the newly formed epiblast epithelium. Subsequent studies have revealed that bone morphogenetic protein 2 (BMP-2) is highly expressed in the endoderm and that expression of a dominant-negative (DN) BMP receptor in EBs blocked cavitation, suggesting BMP-2 to be a death factor (6). The survival signals from the interaction of the epiblast cells with the BM were studied by treating the EBs with polyclonal antiserum against membrane glycoproteins consisting of ECM adhesion receptors. The antiserum treatment induced programmed cell death in the BM-contacting epiblast layer. However, the identities of the receptors and the downstream signaling molecules involved have not been explored.In this study, we utilized EBs differentiated from genetically modified ES cells to investigate the mechanisms of BM-dependent cell survival. We show that targeted deletion of the Rac1 gene in EBs leads to massive apoptosis of epiblast cells in contact with the BM. Rac1 is activated in a BM- and integrin-dependent fashion. Stable expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 also blocks the apoptosis of inner cells and cavitation. These results suggest that the spatial activation of Rac1 is essential not only for BM-dependent epiblast survival but also for apoptosis-mediated cavitation. We further show that Crk mediates Rac1 activation by recruiting the Rac1-specific activator DOCK180 to the cell-BM adhesions and that the phosphatidylinositol 3-kinase (PI3K)-Akt pathway acts downstream of Rac1 to promote BM-dependent survival. Collectively, our results have established a key role for Rac1 in embryonic epithelial morphogenesis and have uncovered a signaling pathway that mediates BM-dependent epithelial survival.     

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels^1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals⁴. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation⁷. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor⁸ and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications^9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls^12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood¹⁵. Analyzing the lignin Raman bands^16,17 in the spectral region between 1,600 and 1,700 cm^-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-μm¹⁸. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.Download video file.^{(47M, mov)}     

On computational approaches for size-and-shape distributions from sedimentation velocity analytical ultracentrifugation

Sedimentation velocity analytical ultracentrifugation has become a very popular technique to study size distributions and interactions of macromolecules. Recently, a method termed two-dimensional spectrum analysis (2DSA) for the determination of size-and-shape distributions was described by Demeler and colleagues (Eur Biophys J 2009). It is based on novel ideas conceived for fitting the integral equations of the size-and-shape distribution to experimental data, illustrated with an example but provided without proof of the principle of the algorithm. In the present work, we examine the 2DSA algorithm by comparison with the mathematical reference frame and simple well-known numerical concepts for solving Fredholm integral equations, and test the key assumptions underlying the 2DSA method in an example application. While the 2DSA appears computationally excessively wasteful, key elements also appear to be in conflict with mathematical results. This raises doubts about the correctness of the results from 2DSA analysis.