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- Comparative phosphoproteomic analysis of checkpoint recovery identifies new regulators of the DNA damage response. [Journal Article, Research Support, Non-U.S. Gov't]
- Sci Signal 2013 Apr 23; 6(272):rs9.
How cells recover from a DNA damage-induced arrest is currently poorly understood. We performed large-scale quantitative phosphoproteomics to identify changes in protein phosphorylation that occurred during recovery from arrest in the G2 phase of the cell cycle caused by DNA damage. We identified 154 proteins that were differentially phosphorylated, and systematic depletion of each of these differentially phosphorylated proteins by small interfering RNA (siRNA) identified at least 10 potential regulators of recovery. Astrin, a protein associated with the mitotic spindle, was among the potential regulators of recovery. We found that astrin controlled the abundance of the cell cycle regulator p53 during DNA damage-induced arrest. Cells in which astrin was depleted had decreased murine double minute 2 (MDM2) abundance and increased p53 at the later stages of the DNA damage response. Astrin was required for continued expression of genes encoding proteins that promote cell cycle progression in arrested cells. Thus, by controlling p53 abundance in cells recovering from DNA damage, astrin maintains the cells in a state competent to resume the cell cycle.
- The microtubule-associated protein astrin transgenesis rescues spermatogenesis and renal function in hypogonadic (hgn/hgn) rats. [Journal Article, Research Support, Non-U.S. Gov't]
- Andrology 2013 Mar; 1(2):301-7.
Male hypogonadic (hgn/hgn) rats show male sterility, reduced female fertility, progressive renal insufficiency and body growth retardation. These defects are associated with loss-of-function mutation of astrin and appear to be related to organ hypoplasia resulting from abnormal cell proliferation and increased cell death during embryonic and early postnatal development. As targeted disruption of mouse spag5 (astrin ortholog) has been reported to show no phenotype, we performed rescue experiments based on the introduction of rat astrin cDNA transgene into hgn/hgn rats to determine whether astrin is actually necessary for the establishment of normal male fertility and renal function. Astrin transgenic (Tg) rats were mated with hgn/+ rats of the HGN strain, and Tg-hgn/+ rats were then crossed to obtain Tg-hgn/hgn. Tg-hgn/hgn males showed recovery of body growth, fertility and renal function. Testis size was smaller in these transgenic animals than normal controls, but showed an increase by 16.5-fold compared with hgn/hgn males. Spermatogenesis occurred in Tg-hgn/hgn testes, and their accessory reproductive organs were of approximately normal size. hgn/hgn males show hypergonadotropic hypogonadism. Increased testosterone and decreased LH levels in Tg-hgn/hgn serum indicated the recovery of Leydig cells' function. Tg-hgn/hgn males showed normal reproductive behaviour, and their mating with Tg-hgn/hgn females produced pups in normal litter size. Their renal sizes and glomerular numbers showed complete recovery, and renal function assayed by biochemical parameters was normal. These results indicated that the transgene is functional in the testis and kidney development as well as body growth. In conclusion, astrin is necessary for the establishment of normal size (cell number) and function of the testis and kidney in rats.
- The mitotic spindle protein SPAG5/Astrin connects to the Usher protein network postmitotically. [Journal Article]
- Cilia 2012; 1(1):2.
ABSTRACT:Mutations in the gene for Usher syndrome 2A (USH2A) are causative for non-syndromic retinitis pigmentosa and Usher syndrome, a condition that is the most common cause of combined deaf-blindness. To gain insight into the molecular pathology underlying USH2A-associated retinal degeneration, we aimed to identify interacting proteins of USH2A isoform B (USH2AisoB) in the retina.We identified the centrosomal and microtubule-associated protein sperm-associated antigen (SPAG)5 in the retina. SPAG5 was also found to interact with another previously described USH2AisoB interaction partner: the centrosomal ninein-like protein NINLisoB. Using In situ hybridization, we found that Spag5 was widely expressed during murine embryonic development, with prominent signals in the eye, cochlea, brain, kidney and liver. SPAG5 expression in adult human tissues was detected by quantitative PCR, which identified expression in the retina, brain, intestine, kidney and testis. In the retina, Spag5, Ush2aisoB and NinlisoB were present at several subcellular structures of photoreceptor cells, and colocalized at the basal bodies.Based on these results and on the suggested roles for USH proteins in vesicle transport and providing structural support to both the inner ear and the retina, we hypothesize that SPAG5, USH2AisoB and NINLisoB may function together in microtubule-based cytoplasmic trafficking of proteins that are essential for cilium formation, maintenance and/or function.
- Exploring diversity in cryptorhynchine weevils (Coleoptera) using distance-, character- and tree-based species delineation. [Journal Article, Research Support, Non-U.S. Gov't]
- Mol Phylogenet Evol 2012 Apr; 63(1):1-14.
Species boundaries are studied in a group of beetles, the western Palaearctic Cryptorhynchinae. We test for congruence of 'traditionally' identified morphospecies with species inferred through parsimony networks, distance-based clustering and the ultrametric tree-based generalized mixed yule-coalescent (GMYC) approach. For that purpose, we sequenced two variable fragments of mitochondrial DNA (CO1 and 16S) for a total of 791 specimens in 217 species of Cryptorhynchinae. Parsimony networks, morphology-calibrated distance clusters and the different tree-based species inferences all achieved low congruence with morphospecies, at best 60%. Although the degree of match with morphospecies was often similar for the different approaches, the composition of clusters partially varied. A barcoding gap was absent in morphospecies-oriented distances as well as for GMYC species clusters. This demonstrates that not only erroneous taxonomic assignments, incomplete lineage sorting, hybridization, or insufficient sampling can compromise distance-based identification, but also differences in speciation rates and uneven tree structure. The initially low match between morphospecies and the different molecular species delineation methods in this case study shows the necessity of combining the output of various methods in an integrative approach. Thereby we obtain an idea about the reliability of the different results and signals, which enables us to fine-tune sampling, delineation technique and data collection, and to identify species that require taxonomic revision.
- The astrin-kinastrin/SKAP complex localizes to microtubule plus ends and facilitates chromosome alignment. [Journal Article, Research Support, Non-U.S. Gov't]
- J Cell Biol 2011 Mar 21; 192(6):959-68.
Astrin is a mitotic spindle-associated protein required for the correct alignment of all chromosomes at the metaphase plate. Astrin depletion delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset. Here we describe an astrin complex containing kinastrin/SKAP, a novel kinetochore and mitotic spindle protein, and three minor interaction partners: dynein light chain, Plk1, and Sgo2. Kinastrin is the major astrin-interacting protein in mitotic cells, and is required for astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Cells overexpressing or depleted of kinastrin mislocalize astrin and show the same mitotic defects as astrin-depleted cells. Importantly, astrin fails to localize to and track microtubule plus ends in cells depleted of or overexpressing kinastrin. These findings suggest that microtubule plus end targeting of astrin is required for normal spindle architecture and chromosome alignment, and that perturbations of this pathway result in delayed mitosis and nonphysiological separase activation.
- Aurora B kinase controls the targeting of the Astrin-SKAP complex to bioriented kinetochores. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- J Cell Biol 2010 Oct 18; 191(2):269-80.
During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B-dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin-SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint-dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores.
- CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore-microtubule dynamics to promote mitotic progression and fidelity. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- EMBO J 2010 Oct 20; 29(20):3531-43.
Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore-microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b-CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin-CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1-astrin-Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.
- Molecular species identification of Central European ground beetles (Coleoptera: Carabidae) using nuclear rDNA expansion segments and DNA barcodes. [Journal Article]
- Front Zool 2010.:26.
The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous.We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae.Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.
- Astrin regulates meiotic spindle organization, spindle pole tethering and cell cycle progression in mouse oocytes. [Journal Article, Research Support, Non-U.S. Gov't]
- Cell Cycle 2009 Oct 15; 8(20):3384-95.
Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrest. Thr24, Ser66 or Ser447 may be the potential phosphorylation sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at metaphase I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.
- SNM1B/Apollo interacts with astrin and is required for the prophase cell cycle checkpoint. [Journal Article, Research Support, N.I.H., Extramural]
- Cell Cycle 2009 Feb 15; 8(4):628-38.
Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-beta-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.