Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently from centrosomes, and the molecular requirements of this pathway remain to be understood. Here we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. Firstly, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation

Mitotic phosphatases: no longer silent partners

Recent work has highlighted the important role played by protein phosphatase complexes in the regulation of mitosis from yeast to mammals. There have been important advances in defining the roles of the protein serine/threonine phosphatases PP1 and PP2A and the dual specificity protein tyrosine phosphatases CDC25 and Cdc14. Three independent studies defined a regulatory role for PP2A in the control of sister chromatid cohesion, involving a direct interaction with shugoshin. A chromatin targeting subunit has been identified for PP1 and the complex shown to play an essential role in chromosome segregation. Key regulatory residues within CDC25 have been mapped and its activity tied both to the initial activation of cyclin-dependent kinases at the centrosome and to DNA damage checkpoints. Novel roles have been defined for Cdc14, including regulation of rDNA and telomere segregation and participation in spindle assembly. These exciting advances show that protein phosphatases are not merely silent partners to kinases in regulating the control of cell division. Introduction The process of cell division is complex and involves multiple independent regulatory steps, most of which are controlled by reversible protein phosphorylation. In higher eukaryotes, mitosis involves condensation of chromosomes, disassembly of the nuclear lamina, breakdown of the nuclear envelope and disassembly of many forms of nuclear bodies, including nucleoli. Completion of mitosis requires alignment and proper segregation of chromosomes into daughter cells followed by reassembly of nuclei and cytokinesis. These and many other events, such as centrosome separation and spindle assembly, are tightly regulated, and several critical checkpoints occur during mitosis to ensure fidelity. Failure to complete any of the key steps can trigger apoptosis and cell death. While the important role of protein phosphorylation in regulating mitotic events has long been recognized, much of the work in this area has focused on the kinases, primarily the Cdk/Cyclin, Aurora, Polo-like and NIMA families (see This review will focus on recent advances in understanding the contributions of four major classes of protein phosphatases to the regulation of processes involved in controlling cell division, specifically the protein serine/ threonine phosphatases PP1 and PP2A and the dualspecificity protein tyrosine phosphatases (DUSPs) CDC25 and Cdc14. We will draw on examples from species as diverse as yeast, insects and mammals, reflecting the high evolutionary conservation of these regulated events. Serine/threonine phosphatases Both PP1 (termed Glc7 in budding yeast and Dis2 in fission yeast) and PP2A are serine/threonine-specific protein phosphatase catalytic subunits that form holoenzyme complexes with one or more regulatory subunits. These regulatory subunits can affect cellular location and/or substrate specificity. In contrast with most kinases, the PP1 and PP2A catalytic subunits can potentially act on a wide range of substrates and thus substrate specificity is conferred by their interaction partners. Thus, a critical step in understanding the role of PP1 and PP2A holoenzymes is to define their regulatory subunits and the mechanism by which they are targeted to their physiological substrates. Much of the literature ascribing specific roles to PP1 or PP2A has relied on differential effects of inhibitors such as okadaic acid, which in vitro blocks PP2A activity at lower concentrations than are required to inhibit PP1 PP2A PP2A plays a prominent role in the regulation of mitosis and signalling pathways. In addition to its interaction with both scaffolding and variable subunits (termed 'A' and 'B' subunits, respectively) in a trimeric complex (see Using immunoprecipitation and yeast two-hybrid studies, several groups independently identified a specific PP2A trimeric complex that interacts with Sgo1 [7 ,8 ,9 ]. On the basis of RNAi studies and analysis of a non-PP2A-binding hSgo1 mutant, Tang and colleagues [7 ] proposed that interaction with PP2A is required for centromeric localization of hSgo1 and proper chromosome segregation. Independently, the same PP2A complex was immunopurified from HEK 293T cells using Flagtagged hSgo1 [8 ]. Immunofluorescence studies by Kitajima and colleagues showed colocalization of hSgo1 and the B56 PP2A regulatory subunit at mammalian centromeres. Using RNAi in mammals, they also reported that knockdown of hSgo2, but not of hSgo1, resulted in loss of centromeric PP2A. Conversely, knockdown of PP2A led to a loss of centromeric hSgo1 [8 ]. Studies on both budding and fission yeast undergoing meiosis also showed that Sgo1 interacts with PP2A at centromeres and serves to protect the cohesin Rec8 subunit from phosphorylation and cleavage [9 ]. Interestingly, tethering of yeast PP2A at specific sites on chromosome arms preserved cohesion at these sites even after meiosis I, when arm cohesin should dissociate, showing an intrinsic ability of PP2A to protect cohesin, independent of Sgo1 [8 ,9 ]. The PP2A complex may thus work both directly at centromeres to maintain cohesion and by facilitating accumulation of Sgo1, which also acts to prevent cleavage of cohesin. Taken together, these studies point to an important new role for PP2A in the control of chromosome cohesion, mediated, at least in part, through interactions with shugoshins ( PP2A has also been implicated in regulating mitotic exit. Wang and Ng [10] provided evidence suggesting that a PP2A-Cdc55 complex dephosphorylates the mitotic exit network (MEN) activator Tem1 in budding yeast. This prevents mitotic exit by blocking release of Cdc14 from 624 Cell division, growth and death Figure 1 Role of PP2A in maintenance of chromosome cohesion. This diagram summarizes three recent studies that identified a specific PP2A trimeric complex acting with shugoshin to protect cohesin at centromeres from phosphorylation and cleavage until the metaphase-anaphase transition. In metazoan mitosis, cohesin is removed from chromosome arms at prometaphase but remains at the centromere regions, protected by shugoshin and PP2A. At the metaphase-anaphase transition, separase is activated and cleaves this residual cohesin, resulting in a loss of cohesion and separation of sister chromatids. PP1 PP1 has been shown to contribute to the regulation of multiple cellular processes including glycogen metabolism and muscle contraction, mediated by interaction of the PP1 catalytic domain with regulatory proteins termed 'targeting subunits'. Over 50 have been described to date, and they have the potential to regulate both the localization and the catalytic activity of PP1 (see [14] for review). Most targeting subunits share a common 'RVXF' motif that mediates direct binding to PP1 An elegant series of experiments has described a role for PP1 in controlling nuclear envelope assembly at the end of mitosis [24][25][26]. When cells enter mitosis, nuclear lamina disassembly is promoted by phosphorylation of B-type lamins. AKAP149, an ER and nuclear membrane protein, was shown to target PP1 (via an RVXF motif) to dephosphorylate B-type lamins at telophase, enabling their polymerization and thus lamina reassembly. A short peptide from AKAP149 containing the RVXF motif can displace PP1 and induce mislocalization of B-type lamins to the cytoplasm. Although the cells were able to complete mitosis, they died by apoptosis within six hours, suggesting that disruption of lamin assembly may directly trigger apoptosis. The association of PP1 isoforms with centrosomes, kinetochores and the cellular cortex and midbody region (see Pinsky et al. [29 ] took advantage of the regulation of Glc7 by targeting subunits to explore its interaction with Ipl1 (Aurora B) in budding yeast. Glc7 is known to antagonize Ipl1 activity, but it was unclear whether it dephosphorylates its substrates or regulates the kinase directly. Although the targeting subunit has not been identified, titratation of Glc7 away from Ipl1 by overexpression of Glc7 binding proteins that do not play roles in chromosome segregation led to increased phosphorylation of Ipl1 substrates, leading the authors to propose that Glc7 acts to ensure accurate chromosome segregation by dephosphorylating Ipl1 targets. CDC25 CDC25 was first identified in fission yeast 20 years ago as a factor required for entry into mitosis [30]. It is now known to activate cyclin-dependent kinases (Cdks) by removing inhibitory phosphates, which leads to Cdk phosphorylation of multiple substrates that drive the cell division process forward (see Three mammalian genes were identified that complement the yeast cdc25 knockout strain. The proteins encoded by these genes, termed CDC25A, CDC25B and CDC25C, are 60% identical in their C-terminal regions, which include the catalytic core containing the CX 5 R motif common to all protein tyrosine phosphatases. In contrast to the reasonably high homology of their catalytic domains, they are only 20-25% identical in their N-terminal regulatory domains, which contain sites for various post-translational modifications and sitespecific protein interactions, including phosphorylation of key serine and threonine residues, ubiquitination, phosphorylation-dependent binding of 14-3-3 proteins and Pin1-dependent prolyl isomerization (see There is a dramatic hyperphosphorylation of CDC25 during the transition from interphase to mitosis, and several mitotic phosphorylation sites have been mapped (see While all three mammalian CDC25 phosphatases activate their Cdk substrates in the same manner, they appear to have distinct roles in regulating cell cycle transitions (see Mitotic phosphatases: no longer silent partners Trinkle-Mulcahy and Lamond 627 have not yet been ruled out. (b) The G 2 /M transition is regulated in a similar way, with CDC25 activating Cdk1/Cyclin B by dephosphorylating critical residues. All three mammalian CDC25 isoforms have been implicated in regulation of this pathway. (c) The initial activation of Cdk1/Cyclin B has been shown to occur at centrosomes as they begin to separate during prophase, and involves the phosphorylation and activation of CDC25B by the Ajuba-Aurora A complex. The divergent N-terminal regulatory domains of the three mammalian CDC25 isoforms contain a variety of regulatory sites, including phosphorylation sites, 14-3-3 binding sites, domains that regulate degradation and nuclear import and export signals. Several of these known and recently described regulatory sites have been summarized here for (d) CDC25A, (e) CDC25B and (f) CDC25C. Cdc14 While Cdc25 is a key regulator of initiation of mitosis (and hence DNA damage checkpoint control), Cdc14 is a key regulator of late mitotic events, coordinating the temporal and spatial control of chromosome segregation with mitotic spindle disassembly and cytokinesis. In the budding yeast S. cerevisiae, Cdc14p plays a key role in exit from mitosis by dephosphorylating Cdk targets (reviewed in FEAR-controlled release of Cdc14p in budding yeast is also important for division of nucleoli and resolution of highly repetitive rDNA and telomere regions, as demonstrated in two recent studies. These regions separate at mid-anaphase, long after cohesin is cleaved. D'Amours and colleagues 628 Cell division, growth and death Figure 4 Cross-species comparison of Cdc14 localization and function. Cdc14 homologues from four different eukaryotes are listed, showing their localization during interphase and throughout mitosis. Nuclei are shown in green, spindle pole bodies (centrosomes) in red, microtubules in pink and chromosomes in blue. The localization of Cdc14 at these sites is shown in yellow. Known mitotic functions for these homologues are also listed

Inner nuclear membrane proteins : targeting and influence on genome organization

The nuclear envelope is a complex double membrane system that separates the activities of the nuclear and cytoplasmic compartments. A recent explosion in the number of proteins associated with this subnuclear organelle together with it now being linked to over 2 dozen diseases indicates the importance of better understanding its functional organisation. This thesis addresses two important questions for this: how do integral proteins of the nuclear envelope get to their sites of function and do any of these proteins direct genome organisation? To address the first question I used FRAP and photoactivation methods to find that different proteins use at least 4 distinct mechanisms to reach the inner nuclear membrane. Some appeared to be translocated by simple unaided lateral diffusion in the membrane while others needed Ran GTPase activity, others ATP, some others were aided by phenylalanine/glycines (FGs). Both Ran and FG mechanisms required the nucleoporin Nup35, albeit the mechanisms appeared to be completely independent of one another. To investigate the role of the nuclear envelope in genome organization, I screened for nuclear envelope proteins that reposition particular chromosomes to the nuclear periphery, finding five with this function. Interestingly, all of the proteins with this effect are tissuespecific. Depletion of two liver-specific nuclear envelope proteins reversed their effects on a specific chromosome for positioning with respect to the nuclear periphery. Finally, exogenous expression of these proteins in tissue culture cells caused induction of genes involved in differentiation pathways.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Earth System Model Evaluation Tool (ESMValTool) v2.0-technical overview

This paper describes the second major release of the Earth System Model Evaluation Tool (ESMValTool), a community diagnostic and performance metrics tool for the evaluation of Earth system models (ESMs) participating in the Coupled Model Intercomparison Project (CMIP). Compared to version 1.0, released in 2016, ESMValTool version 2.0 (v2.0) features a brand new design, with an improved interface and a revised preprocessor. It also Features a significantly enhanced diagnostic part that is described in three companion papers. The new version of ESMValTool has been specifically developed to target the increased data volume of CMIP Phase 6 (CMIP6) and the related challenges posed by the analysis and the evaluation of Output from multiple high-resolution or complex ESMs. The new version takes advantage of state-of-the-art computational libraries and methods to deploy an efficient and user-friendly data processing. Common operations on the input data (such as regridding or computation of multi-model statistics) are centralized in a highly optimized preprocessor, which allows applying a series of preprocessing functions before diagnostics scripts are applied for in-depth scientific analysis of the model output. Performance tests conducted on a set of standard diagnostics show that the new version is faster than ist predecessor by about a factor of 3. The performance can be further improved, up to a factor of more than 30, when the newly introduced task-based parallelization options are used, which enable the efficient exploitation of much larger computing infrastructures. ESMValTool v2.0 also includes a revised and simplified installation procedure, the setting of user-configurable options based on modern language formats, and high code quality standards following the best practices for software development

The Speed of Sound and Attenuation of an IEC Agar-Based Tissue-Mimicking Material for High Frequency Ultrasound Applications

This study characterized the acoustic properties of an International Electromechanical Commission (IEC) agar-based tissue mimicking material (TMM) at ultrasound frequencies in the range 10–47 MHz. A broadband reflection substitution technique was employed using two independent systems at 21°C ± 1°C. Using a commercially available preclinical ultrasound scanner and a scanning acoustic macroscope, the measured speeds of sound were 1547.4 ± 1.4 m∙s−1 and 1548.0 ± 6.1 m∙s−1, respectively, and were approximately constant over the frequency range. The measured attenuation (dB∙cm−1) was found to vary with frequency f (MHz) as 0.40f + 0.0076f2. Using this polynomial equation and extrapolating to lower frequencies give values comparable to those published at lower frequencies and can estimate the attenuation of this TMM in the frequency range up to 47 MHz. This characterisation enhances understanding in the use of this TMM as a tissue equivalent material for high frequency ultrasound applications

ESMValCore

ESMValCore: A community tool for pre-processing data from Earth system models in CMIP and running analysis scripts

ESMValCore

ESMValCore: A community tool for pre-processing data from Earth system models in CMIP and running analysis scripts

ESMValCore

ESMValCore: A community tool for pre-processing data from Earth system models in CMIP and running analysis scripts

ESMValTool

ESMValTool: A community diagnostic and performance metrics tool for routine evaluation of Earth system models in CMIP