Role of the Mad2 Dimerization Interface in the Spindle Assembly Checkpoint Independent of Kinetochores

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Highlights? Mad2 overexpression activates the SAC independently from Mad1 in budding yeast ? Mad2 dimerization surface is required for the SAC independently from Mad1 ? Mad2 dimerization downstream of kinetochores is negligible for the SAC ? Mad2 dimerization surface interacts with Mad3 and is required for MCC stability     

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.     

Multiple interactions of FbsA, a surface protein from Streptococcus agalactiae, with fibrinogen: affinity, stoichiometry, and structural characterization

Streptococcus agalactiae is an etiological agent of several infective diseases in humans. We previously demonstrated that FbsA, a fibrinogen-binding protein expressed by this bacterium, elicits a fibrinogen-dependent aggregation of platelets. In the present communication, we show that the binding of FbsA to fibrinogen is specific and saturable, and that the FbsA-binding site resides in the D region of fibrinogen. In accordance with the repetitive nature of the protein, we found that FbsA contains multiple binding sites for fibrinogen. By using several biophysical methods, we provide evidence that the addition of FbsA induces extensive fibrinogen aggregation and has noticeable effects on thrombin-catalyzed fibrin clot formation. Fibrinogen aggregation was also found to depend on FbsA concentration and on the number of FbsA repeat units. Scanning electron microscopy evidentiated that, while fibrin clot is made of a fine fibrillar network, FbsA-induced Fbg aggregates consist of thicker fibers organized in a cage-like structure. The structural difference of the two structures was further indicated by the diverse immunological reactivity and capability to bind tissue-type plasminogen activator or plasminogen. The mechanisms of FbsA-induced fibrinogen aggregation and fibrin polymerization followed distinct pathways since Fbg assembly was not inhibited by GPRP, a specific inhibitor of fibrin polymerization. This finding was supported by the different sensitivity of the aggregates to the disruptive effects of urea and guanidine hydrochloride. We suggest that FbsA and fibrinogen play complementary roles in contributing to thrombogenesis associated with S. agalactiae infection.     

Mitigating human disturbance: can protection influence trajectories of recovery in benthic assemblages?

1. Understanding whether Marine Protected Areas (MPAs) can be considered as a suitable tool for restoring the structure and function of populations and assemblages is urgently needed to achieve an effective policy of mitigation of human impact in coastal management. However, to date, the role played by MPAs in enhancing ecosystems resilience has been more advocated than unambiguously documented. 2. This study was designed to test whether full protection in marine reserves facilitates recovery of benthos impacted by the date mussel Lithophaga lithophaga fishery, one of the most harmful human activities affecting subtidal rocky habitats in the Mediterranean Sea. 3. The effects of this destructive fishery were reproduced at one fully protected location (P) and at two unprotected control locations (Cs) in the SW Mediterranean Sea. At each location, three plots (4 m2) of rocky surface at 4-6 m depth were disturbed experimentally, while another three plots served as reference. In each plot, the species composition and relative cover of the sessile benthic assemblages were sampled photographically on each of five occasions during a period of 20 months. 4. Over and above variation in habitat features among locations, multivariate and univariate analyses revealed significant differences between P-vs.-Cs in patterns of assemblage recovery and showed that, at the fully protected location, recovery was faster than at the unprotected control locations. 5. Our results suggest that MPAs have the potential to change the trajectories of recovery of disturbed assemblages by accelerating the processes of recolonization and call for further investigation to identify the specific mechanisms underlying increased resilience.     

Magnetic resonance imaging of tumor cells by targeting the amino acid transport system

An early diagnosis of cancer is crucial in the battle against this disease and the in vivo visualization of tumors at cellular level is still the most challenging goal. In order to target tumor cells, we took into account their increased metabolism and amino acid nutrients or pseudo-nutrients, which are actively transported through the cell membrane, have been chosen as vectors for new MRI contrast agents. For this reason new gadolinium complexes conjugated to agmatine, arginine, and glutamine have been synthesized and studied.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

<Emphasis Type="Italic">Anisakis nascettii</Emphasis> n. sp. (Nematoda: Anisakidae) from beaked whales of the southern hemisphere: morphological description,genetic relationships between congeners and ecological data

A new anisakid nematode, Anisakis nascettii n. sp., is described from beaked whales Mesoplodon spp. off the coast of New Zealand and South Africa. Morphological and molecular (allozymes and mtDNA cox2 sequence) data were used for diagnostic and identification purposes. Among the 19 allozymes studied, 10 were found to be unique and characteristic for A. nascettii n. sp. Analysis of allozymes demonstrated reproductive isolation from A. ziphidarum Paggi, Nascetti, Webb, Mattiucci, Cianchi & Bullini, 1998 and mtDNA cox2 sequences depict this Anisakis species as a distinct and unique entity. Key morphological diagnostic traits for A. nascettii with respect to the genetically closely related species A. ziphidarum include: spicule length, the spicule/body length ratio, the arrangement of the caudal papillae and the shape of the plectanes of the adult males. Genetic data confirmed that Anisakis sp. A of Pontes et al. (2005), which was partly described by Iglesias et al. (2008), and Anisakis sp. of Valentini et al. (2006) are conspecific with A. nascettii. Both molecular and morphological data indicate that the new species belongs to the ‘ziphidarum-group’; however, it is genetically very distinct from A. ziphidarum (D _Nei  = 0.69, K2P = 0.09), as well as from all of the previously genetically characterised Anisakis spp. All tree topologies inferred by different methods (MP, NJ and Bayesian) support the finding that A. nascettii n. sp. and A. ziphidarum are sister-species. It is also confirmed that A. nascettii n. sp. is, at the adult stage, a parasite of beaked whales of the genus Mesoplodon, whereas, as a larva, it has been identified from the squid Moroteuthis ingens Smith. Furthermore, Mesoplodon bowdoini Andrews represents a new host record for A. ziphidarum. The parallelism between the clade formed by these two anisakine taxa, i.e. A. ziphidarum and A. nascettii, and that formed by their definitive hosts further supports the hypothesis of host–parasite co-evolutionary relationships, as previously suggested for Anisakis spp. and their cetacean hosts.     

Cdc14 Inhibition by the Spindle Assembly Checkpoint Prevents Unscheduled Centrosome Separation in Budding Yeast

The spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance mechanism that delays anaphase onset and mitotic exit in response to the lack of kinetochore attachment. The target of the SAC is the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its Cdc20 activator. The Cdc20/APC complex is in turn required for sister chromatid separation and mitotic exit through ubiquitin-mediated proteolysis of securin, thus relieving inhibition of separase that unties sister chromatids. Separase is also involved in the Cdc-fourteen early anaphase release (FEAR) pathway of nucleolar release and activation of the Cdc14 phosphatase, which regulates several microtubule-linked processes at the metaphase/anaphase transition and also drives mitotic exit. Here, we report that the SAC prevents separation of microtubule-organizing centers (spindle pole bodies [SPBs]) when spindle assembly is defective. Under these circumstances, failure of SAC activation causes unscheduled SPB separation, which requires Cdc20/APC, the FEAR pathway, cytoplasmic dynein, and the actin cytoskeleton. We propose that, besides inhibiting sister chromatid separation, the SAC preserves the accurate transmission of chromosomes also by preventing SPBs to migrate far apart until the conditions to assemble a bipolar spindle are satisfied.