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A novel role for microglia in minimizing excitotoxicity
Microglia are the abundant, resident myeloid cells of the central nervous system (CNS) that become rapidly activated in response to injury or inflammation. While most studies of microglia focus on this phenomenon, little is known about the function of 'resting' microglia, which possess fine, branching cellular processes. Biber and colleagues, in a recent paper in Journal of Neuroinflammation, report that ramified microglia can limit excitotoxicity, an important insight for understanding mechanisms that limit neuron death in CNS disease.See research articlehttp://www.jneuroinflammation.com/content/9/1/27.
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Q&A: What is a pathogen? A question that begs the point
{No abstract}
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Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway
Background:
Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates. The kinase plays important role in a number of cellular processes. The cellular functions of Plasmodium falciparum protein kinase CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK21 and PfCK22.
Results:
We now show that the genes encoding both regulatory PfCK2 subunits, PfCK2beta1 and PfCK2beta2, cannot be disrupted. By examining intra-erythrocytic stages of transgenic parasite lines expressing HA-tagged catalytic and regulatory subunits (HA-CK2alpha, HA-PfCK2beta1 or HA-PfCK2beta2) by immunofluorescence and electron microscopy, we localize all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2beta1- and PfCK2beta2-containing complexes, which were analyzed by mass spectrometry. Recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2beta1 and PfCK2beta2 precipitates, implicating PfCK2 in chromatin dynamics. We further demonstrate that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are indeed phosphorylated by PfCK2alpha in vitro.
Conclusions:
Our reverse genetics data demonstrate that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.
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Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth
Background:
During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.
Results:
To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.
Conclusions:
This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels, and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.
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Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum) lines
Background:
Alteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.
Results:
Multi-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA) and Aegilops tauschii (2n = 2x = 14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.
Conclusions:
Our results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.
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Protein dynamics and conformational selection in bidirectional signal transduction
Protein conformational dynamics simultaneously allow promiscuity and specificity in binding. The multiple conformations of the free EphA4 ligand-binding domain observed in two new EphA4 crystal structures provide a unique insight into the conformational dynamics of EphA4 and its signaling pathways. The heterogeneous ensemble and loop dynamics explain how the EphA4 receptor is able to bind multiple A- and B-ephrin ligands and small molecules via conformational selection, which helps to fine-tune cellular signal response in both receptor and ligand cells.See research article http://www.biomedcentral.com/2046-1682/5/2
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Detailed interrogation of trypanosome cell biology via
differential organelle staining and automated image analysis
Background:
Many trypanosomatid protozoa are important human or animal pathogens. The well-defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analysing the effect of mutations, chemical insults and changes between lifecycle stages. High throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method.
Results:
We have developed a technique based on double staining of the DNA with a minor groove binding (DAPI) and a base pair intercalating (propidium iodide or SYBR green) fluorescent stain and colour deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically.
Conclusions:
Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.
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Difficult phylogenetic questions: more data, maybe; better methods, certainly
Contradicting the prejudice that endosymbiosis is a rare phenomenon, Husník and co-workers show in BMC Biology that bacterial endosymbiosis has occured several times independently during insect evolution. Rigorous phylogenetic analyses, in particular using complex models of sequence evolution and an original site removal procedure, allow this conclusion to be established after eschewing inference artefacts that usually plague the positioning of highly divergent endosymbiont genomic sequences.See research article http://www.biomedcentral.com/1741-7007/9/87
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The bending of cell sheets - from folding to rolling
The bending of cell sheets plays a major role in multicellular embryonic morphogenesis. Recent advances are leading to a deeper understanding of how the biophysical properties and the force-producing behaviors of cells are regulated, and how these forces are integrated across cell sheets during bending. We review work that shows that the dynamic balance of apical versus basolateral cortical tension controls specific aspects of invagination of epithelial sheets, and recent evidence that tissue expansion by growth contributes to neural retinal invagination in a stem cell-derived, self-organizing system. Of special interest is the detailed analysis of the type B inversion in Volvox reported in BMC Biology by Höhn and Hallmann, as this is a system that promises to be particularly instructive in understanding morphogenesis of any monolayered spheroid system.See research article: http://www.biomedcentral.com/1741-7007/9/89
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There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator
Background:
Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult conguration; this process is called "inversion." There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out?
Results:
In this study, we investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri.
Conclusions:
Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator.
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