- The 2018 Revision of the ISBER Best Practices: Summary of Changes and the Editorial Team's Development Process. [Journal Article]
- BBBiopreserv Biobank 2018; 16(1):3-6
- An increased need for specimens of reliable and consistent quality for research purposes requires the development of standardized policies and practices for the collection, handling, storage, retriev...
An increased need for specimens of reliable and consistent quality for research purposes requires the development of standardized policies and practices for the collection, handling, storage, retrieval, and distribution of specimens and specimen-related data. Providers of specimen resources should strive to incorporate new technologies and state-of-the-science approaches and thus ensure the availability of fit-for-purpose research specimens. Strategies to achieve quality outcomes and performance improvements often include adherence to established standards and implementation of best practices. Although standards represent a rigid set of guidelines that define exactly how a task should be completed, best practices are recommended actions and principles that demonstrate an awareness of standards, solve problems, can be replicated, and work in a given context. Adoption of best practice elements will vary based on the goals and circumstances of a given initiative, and in some instances, may not be possible to implement or may represent an aspirational achievement. In an effort to harmonize the scientific, technical, legal, and ethical issues relevant to repositories of biological and environmental specimens, the International Society for Biological and Environmental Repositories (ISBER) has released the updated ISBER Best Practices: Recommendations for Repositories (ISBER Best Practices). The document provides a comprehensive tool to guide repository professionals in both managerial and technical aspects such as practical details on repository governance, development, and operation; regulatory compliance; and ethical, legal, and social issues relevant to repositories. This summary describes the process for revising the document and summarizes the new topics, updates, and areas of expansion included in the fourth edition of ISBER Best Practices.
- Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80. [Journal Article]
- EElife 2017 Aug 25; 6
- Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attach...
Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.
- Aurora-B kinase pathway controls the lateral to end-on conversion of kinetochore-microtubule attachments in human cells. [Journal Article]
- NCNat Commun 2017 07 28; 8(1):150
- Human chromosomes are captured along microtubule walls (lateral attachment) and then tethered to microtubule-ends (end-on attachment) through a multi-step end-on conversion process. Upstream regulato...
Human chromosomes are captured along microtubule walls (lateral attachment) and then tethered to microtubule-ends (end-on attachment) through a multi-step end-on conversion process. Upstream regulators that orchestrate this remarkable change in the plane of kinetochore-microtubule attachment in human cells are not known. By tracking kinetochore movements and using kinetochore markers specific to attachment status, we reveal a spatially defined role for Aurora-B kinase in retarding the end-on conversion process. To understand how Aurora-B activity is counteracted, we compare the roles of two outer-kinetochore bound phosphatases and find that BubR1-associated PP2A, unlike KNL1-associated PP1, plays a significant role in end-on conversion. Finally, we uncover a novel role for Aurora-B regulated Astrin-SKAP complex in ensuring the correct plane of kinetochore-microtubule attachment. Thus, we identify Aurora-B as a key upstream regulator of end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.Human chromosomes are captured along microtubule walls and then tethered to microtubule-ends through a multi-step end-on conversion process. Here the authors show that Aurora-B regulates end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.
- Critical roles of Astrin in the mitosis of immature rat Sertoli cells. [Journal Article]
- BBBiochem Biophys Res Commun 2017 May 13; 486(4):958-964
- Male hypogonadism (hgn/hgn) rats show testicular hypoplasia accompanied by dysplastic development of seminiferous tubules due to loss-of-function mutation of the gene encoding Astrin, which is requir...
Male hypogonadism (hgn/hgn) rats show testicular hypoplasia accompanied by dysplastic development of seminiferous tubules due to loss-of-function mutation of the gene encoding Astrin, which is required for mitotic progression in the division cycle of HeLa cells. In the present study, we examined the cytological base leading to the decrease of Sertoli cells in hgn/hgn testes. In hgn/hgn testes on postnatal day 3, anti-phospho-histone H3 (Ser10) (pH3)-positive mitotic phase and TUNEL-positive apoptosis increased in GATA4-positive Sertoli cells. Isolated immature Sertoli cells from hgn/hgn testes showed increased pH3-assessed mitotic index accompanied by decreased 5-bromo-2'-deoxyuridine-incorporation and increased TUNEL-positive apoptosis, suggesting mitotic delay and cell death. In the visualization of mitotic progression by nocodazole (NOC)-mediated cell cycle arrest and subsequent release, hgn/hgn rat-derived Sertoli cells failed to make the transition from prometaphase to metaphase, and the cells with micronuclei and TUNEL-positive cells gradually increased in a time-dependent manner. Western blot analysis detected ≈142 kDa protein expected as Astrin in extracts of +/+ and +/hgn testes and cultured normal Sertoli cells but not in extracts of hgn/hgn testes. CLASP1 was detected in extracts of both normal and hgn/hgn testes, whereas it was localized in kinetochore of normal mitotic Sertoli cells but diffused in cytoplasm of hgn/hgn Sertoli cells. These results indicate that Astrin is required for normal mitotic progression in immature Sertoli cells and that the most severe type of testicullar dysplasia in hgn/hgn rats is caused by mitotic cell death of immature Sertoli cells due to lack of Astrin.
- Timing and host plant associations in the evolution of the weevil tribe Apionini (Apioninae, Brentidae, Curculionoidea, Coleoptera) indicate an ancient co-diversification pattern of beetles and flowering plants. [Journal Article]
- MPMol Phylogenet Evol 2017; 107:179-190
- Host plant shifts of insects can lead to a burst of diversification driven by their arrival in a new adaptive zone. In this context, our study aims to explore timing and patterns in the evolution of ...
Host plant shifts of insects can lead to a burst of diversification driven by their arrival in a new adaptive zone. In this context, our study aims to explore timing and patterns in the evolution of the weevil tribe Apionini (Brentidae, Curculionoidea, Coleoptera), particularly in relation to affiliations with their host plants. The classification of Apionini is difficult because of their relatively uniform appearance. Most taxa live mono- or oligophagously on members of Asteraceae or Fabaceae, but many are associated with other plant families, like Lamiaceae, Malvaceae and Polygonaceae. However, a comprehensive hypothesis of the phylogenetic relationships within the tribe Apionini is still missing. In the present study, we reconstructed trees and estimated divergence times among tribes. These results were further used to reconstruct the ancestral host plant use in Apionini weevils and to infer if the divergence timing of putative subtribes corresponds with the occurrence and radiation of their specific host plant groups. Phylogenetic analyses confirm the monophyly of most subtribes, with the exceptions of Oxystomatina, Kalcapiina and Aspidapiina. The subribe Aplemonina is inferred to be sister to all remaining Apionini. Divergence time estimates indicate the first occurrence of Apionini in the Upper Cretaceous and a simultaneous occurrence of several families of flowering plants and the occupation by Apionini weevil herbivores. These conspicuous coincidences support either an ancient co-diversification scenario or an escalating diversification in weevils induced by the radiation of flowering plants.
- Towards a DNA Barcode Reference Database for Spiders and Harvestmen of Germany. [Journal Article]
- PlosPLoS One 2016; 11(9):e0162624
- As part of the German Barcode of Life campaign, over 3500 arachnid specimens have been collected and analyzed: ca. 3300 Araneae and 200 Opiliones, belonging to almost 600 species (median: 4 individua...
As part of the German Barcode of Life campaign, over 3500 arachnid specimens have been collected and analyzed: ca. 3300 Araneae and 200 Opiliones, belonging to almost 600 species (median: 4 individuals/species). This covers about 60% of the spider fauna and more than 70% of the harvestmen fauna recorded for Germany. The overwhelming majority of species could be readily identified through DNA barcoding: median distances between closest species lay around 9% in spiders and 13% in harvestmen, while in 95% of the cases, intraspecific distances were below 2.5% and 8% respectively, with intraspecific medians at 0.3% and 0.2%. However, almost 20 spider species, most notably in the family Lycosidae, could not be separated through DNA barcoding (although many of them present discrete morphological differences). Conspicuously high interspecific distances were found in even more cases, hinting at cryptic species in some instances. A new program is presented: DiStats calculates the statistics needed to meet DNA barcode release criteria. Furthermore, new generic COI primers useful for a wide range of taxa (also other than arachnids) are introduced.
- Nuclear Mitotic Apparatus (NuMA) Interacts with and Regulates Astrin at the Mitotic Spindle. [Journal Article]
- JBJ Biol Chem 2016 09 16; 291(38):20055-67
- The large nuclear mitotic apparatus (NuMA) protein is an essential player in mitotic spindle assembly and maintenance. We report here the identification of Astrin, a spindle- and kinetochore-associat...
The large nuclear mitotic apparatus (NuMA) protein is an essential player in mitotic spindle assembly and maintenance. We report here the identification of Astrin, a spindle- and kinetochore-associated protein, as a novel interactor of NuMA. We show that the C-terminal tail of NuMA can directly bind to the C terminus of Astrin and that this interaction helps to recruit Astrin to microtubules. Knockdown of NuMA by RNA interference dramatically impaired Astrin recruitment to the mitotic spindle. Overexpression of the N terminus of mammalian homologue of Drosophila Pins (LGN), which blocks the microtubule binding of NuMA and competes with Astrin for NuMA binding, also led to similar results. Furthermore, we found that cytoplasmic dynein is required for the spindle pole accumulation of Astrin, and dynein-mediated transport is important for balanced distribution of Astrin between spindle poles and kinetochores. On the other hand, if Astrin levels are reduced, then NuMA could not efficiently concentrate at the spindle poles. Our findings reveal a direct physical link between two important regulators of mitotic progression and demonstrate the critical role of the NuMA-Astrin interaction for accurate cell division.
- Phosphorylation of Astrin Regulates Its Kinetochore Function. [Journal Article]
- JBJ Biol Chem 2016 Aug 19; 291(34):17579-92
- The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic ...
The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.
- How to tackle the molecular species inventory for an industrialized nation-lessons from the first phase of the German Barcode of Life initiative GBOL (2012-2015). [Journal Article]
- GGenome 2016; 59(9):661-70
- Biodiversity loss is mainly driven by human activity. While concern grows over the fate of hot spots of biodiversity, contemporary species losses still prevail in industrialized nations. Therefore, s...
Biodiversity loss is mainly driven by human activity. While concern grows over the fate of hot spots of biodiversity, contemporary species losses still prevail in industrialized nations. Therefore, strategies were formulated to halt or reverse the loss, driven by evidence for its value for ecosystem services. Maintenance of the latter through conservation depends on correctly identified species. To this aim, the German Federal Ministry of Education and Research is funding the GBOL project, a consortium of natural history collections, botanic gardens, and universities working on a barcode reference database for the country's fauna and flora. Several noticeable findings could be useful for future campaigns: (i) validating taxon lists to serve as a taxonomic backbone is time-consuming, but without alternative; (ii) offering financial incentives to taxonomic experts, often citizen scientists, is indispensable; (iii) completion of the libraries for widespread species enables analyses of environmental samples, but the process may not hold pace with technological advancements; (iv) discoveries of new species are among the best stories for the media; (v) a commitment to common data standards and repositories is needed, as well as transboundary cooperation between nations; (vi) after validation, all data should be published online via the BOLD to make them searchable for external users and to allow cross-checking with data from other countries.
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- A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends. [Journal Article]
- JCJ Cell Biol 2016 05 09; 213(3):315-28
- The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP i...
The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning.