64 research outputs found
Luminescence Dating in Fluvial Settings: Overcoming the Challenge of Partial Bleaching
Optically stimulated luminescence (OSL) dating is a versatile technique that utilises the two most ubiquitous minerals on Earth (quartz or K-feldspar) for constraining the timing of sediment deposition. It has provided accurate ages in agreement with independent age control in many fluvial settings, but is often characterised by partial bleaching of individual grains. Partial bleaching can occur where sunlight exposure is limited and so only a portion of the grains in the sample was exposed to sunlight prior to burial, especially in sediment-laden, turbulent or deep water columns. OSL analysis on multiple grains can provide accurate ages for partially bleached sediments where the OSL signal intensity is dominated by a single brighter grain, but will overestimate the age where the OSL signal intensity is equally as bright (often typical of K-feldspar) or as dim (sometimes typical of quartz). In such settings, it is important to identify partial bleaching and the minimum dose population, preferably by analysing single grains, and applying the appropriate statistical age model to the dose population obtained for each sample. To determine accurate OSL ages using these age models, it is important to quantify the amount of scatter (or overdispersion) in the well-bleached part of the partially bleached dose distribution, which can vary between sediment samples depending upon the bedrock sources and transport histories of grains. Here, we discuss how the effects of partial bleaching can be easily identified and overcome to determine accurate ages. This discussion will therefore focus entirely on the burial dose determination for OSL dating, rather than the dose-rate, as only the burial doses are impacted by the effects of partial bleaching
In Vitro Contraction of Cytokinetic Ring Depends on Myosin II but not on Actin Dynamics
10.1038/ncb2781Nature Cell Biology157853-859NCBI
Murine Missing in Metastasis (MIM) Mediates Cell Polarity and Regulates the Motility Response to Growth Factors
Missing in metastasis (MIM) is a member of the inverse BAR-domain protein family, and in vitro studies have implied MIM plays a role in deforming membrane curvature into filopodia-like protrusions and cell dynamics. Yet, the physiological role of the endogenous MIM in mammalian cells remains undefined.We have examined mouse embryonic fibroblasts (MEFs) derived from mice in which the MIM locus was targeted by a gene trapping vector. MIM(-/-) MEFs showed a less polarized architecture characterized by smooth edges and fewer cell protrusions as compared to wild type cells, although the formation of filopodia-like microprotrusions appeared to be normal. Immunofluorescent staining further revealed that MIM(-/-) cells were partially impaired in the assembly of stress fibers and focal adhesions but were enriched with transverse actin filaments at the periphery. Poor assembly of stress fibers was apparently correlated with attenuation of the activity of Rho GTPases and partially relieved upon overexpressing of Myc-RhoA(Q63L), a constitutively activated RhoA mutant. MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum. Upon treatment with PDGF MIM(-/-) cells developed more prominent dorsal ruffles along with increased Rac1 activity. Compared to wild type cells, MIM(-/-) cells had a slower motility in the presence of a low percentage of serum-containing medium but migrated normally upon adding growth factors such as 10% serum, PDGF or EGF. MIM(-/-) cells were also partially impaired in the internalization of transferrin, fluorescent dyes, foreign DNAs and PDGF receptor alpha. On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling
Redistribution of Actin during Assembly and Reassembly of the Contractile Ring in Grasshopper Spermatocytes
Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end–directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules
Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19
Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold
Roles of the DYRK Kinase Pom2 in Cytokinesis, Mitochondrial Morphology, and Sporulation in Fission Yeast
Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast
Coupling changes in cell shape to chromosome segregation
Animal cells undergo dramatic changes in shape, mechanics and polarity as they progress through the different stages of cell division. These changes begin at mitotic entry, with cell–substrate adhesion remodelling, assembly of a cortical actomyosin network and osmotic swelling, which together enable cells to adopt a near spherical form even when growing in a crowded tissue environment. These shape changes, which probably aid spindle assembly and positioning, are then reversed at mitotic exit to restore the interphase cell morphology. Here, we discuss the dynamics, regulation and function of these processes, and how cell shape changes and sister chromatid segregation are coupled to ensure that the daughter cells generated through division receive their fair inheritance
Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling
Comprehensive molecular characterization of the hippo signaling pathway in cancer
Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional “omic” data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era
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