Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

In cytokinesis with chromatin bridges, cells delay abscission and retain actin patches at the intercellular canal to prevent chromosome breakage. In this study, we show that inhibition of Src, a protein-tyrosine kinase that regulates actin dynamics, or Chk1 kinase correlates with chromatin breakage and impaired formation of actin patches but not with abscission in the presence of chromatin bridges. Chk1 is required for optimal localization and complete activation of Src. Furthermore, Chk1 phosphorylates human Src at serine 51, and phosphorylated Src localizes to actin patches, the cell membrane, or the nucleus. Nonphosphorylatable mutation of S51 to alanine reduces Src catalytic activity and impairs formation of actin patches, whereas expression of a phosphomimicking Src-S51D protein rescues actin patches and prevents chromatin breakage in Chk1-deficient cells. We propose that Chk1 phosphorylates Src-S51 to fully induce Src kinase activity and that phosphorylated Src promotes formation of actin patches and stabilizes chromatin bridges. These results identify proteins that regulate formation of actin patches in cytokinesis

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders

BackgroundNeuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in “orphan” AES cases.MethodsThe study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 “control” patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR–transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies’ binding to rat brain tissue.ResultsThree patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient‐derived serum anti‐nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti‐nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti‐nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis.ConclusionThis study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients

Chk1 and Src proteins prevent chromatin bridge breakage in cytokinesis

Chromatin bridges are DNA strings of incompletely segregated chromatin which connect the daughter nuclei during cytokinesis. If unresolved, chromatin bridges can be fragmented during furrow ingression in cytokinesis, leading to genetic instability. The presence of chromatin bridges activates the abscission checkpoint which in turn delays the final cut of the cytoplasm, thus protecting from DNA bridge breakage. Furthermore, actin-rich structures known as actin patches, are formed at the base of the chromatin bridge. It has been suggested that under normal conditions, the actin patches stabilize the intercellular canal until the DNA bridge is resolved. Though, how actin patches are formed remains largely enigmatic. The nonreceptor tyrosine kinase Src, has been implicated in the rearrangement of actin cytoskeleton. In the present study, we have shown that in human cells, Src inhibition leads to increased chromatin bridge breakage and elevated frequency of cells exhibiting micronuclei. However, DNA bridge breakage in Chk1- or Src-deficient cells correlates with reduced actin patches at the bases of chromatin bridges and is not caused by abscission. Activated Src is localized to the actin patches at the bases of DNA bridges during cytokinesis. Additionally, Chk1 downregulation results in reduced Src activity, loss of actin patches and increased DNA bridge breakage. Furthermore, we found that Chk1 phosphorylates Src at a newly identified site, Serine 51, and that phosphorylation is required to fully induce Src kinase activity. Phosphorylated Src at Serine 51 is localized at the bases of DNA bridges, at the cellular membrane and the nucleus. Mutation of Serine 51 to the non-phosphorylatable aminoacid Alanine, leads to reduced actin patches and increased DNA chromatin breakage, whereas expression of a phosphomimicking Src-S51D protein rescues actin patches and prevents chromatin breakage in Chk1-deficient cells. Based on these results, we suggest a novel mechanism required for stabilization of DNA bridges during cytokinesis, which involves Chk1- and Src- dependent formation of actin patches at the bases of chromatin bridges.Οι γέφυρες χρωματίνης είναι νήματα DNA τα οποία ενώνουν τους θυγατρικούς πυρήνες κατά το τέλος της κυτταροκίνησης και, εάν δεν επιλυθούν, μπορούν να σπάσουν προκαλώντας χρωμοσωμική αστάθεια. Παρουσία γεφυρών χρωματίνης ενεργοποιείται το abscission checkpoint το οποίο καθυστερεί το τελικό κόψιμο του κυτταροπλάσματος, για να μη σπάσουν οι γέφυρες χρωματίνης. Ακόμη, εκατέρωθεν των χρωματινικών γεφυρών σχηματίζονται δομές ακτίνης (actin patches), οι οποίες στα φυσιολογικά κύτταρα σταθεροποιούν τις γέφυρες DNA έως ότου αυτές επιλυθούν. Ωστόσο, ο μηχανισμός δημιουργίας των actin patches δεν έχει ακόμα βρεθεί. Η πρωτεϊνική κινάση τυροσίνης Src, συμμετέχει στην αναδιοργάνωση του κυτταροσκελετού. Στην παρούσα μελέτη, δείξαμε ότι αναστολή της Src σε κύτταρα ανθρώπου οδηγεί σε αύξηση του ποσοστού των σπασμένων γεφυρών DNA στην κυτταροκίνηση και αύξηση του ποσοστού των κυττάρων με μικροπυρήνες. Ωστόσο, το σπάσιμο των γεφυρών DNA σε κύτταρα χωρίς λειτουργική Chk1 ή Src συσχετίζεται με απώλεια των actin patches στις βάσεις των γεφυρών DNA και όχι με δυσλειτουργία του abscission checkpoint. Η ενεργοποιημένη πρωτεΐνη Src εντοπίζεται στα actin patches, στις βάσεις των γεφυρών DNA στην κυτταροκίνηση. Επίσης, μείωση της κινάσης Chk1 οδηγεί σε μείωση της ενεργότητας της πρωτεΐνης Src, απώλεια των actin patches και αύξηση του ποσοστού των σπασμένων γεφυρών DNA στην κυτταροκίνηση. Ακόμη, δείξαμε ότι η Chk1 φωσφορυλιώνει την πρωτεΐνη Src σε μία νέα θέση, τη Σερίνη 51 και ότι η φωσφορυλιωμένη Src στη Σερίνη 51 εντοπίζεται στις βάσεις των γεφυρών DNA, την κυτταρική μεμβράνη και τον πυρήνα. Η φωσφορυλίωση της Src στη Σερίνη 51 απαιτείται για πλήρη ενεργοποίηση της Src. Έκφραση της μεταλλαγμένης Src S51A, στην οποία η Σερίνη 51 έχει μεταλλαχθεί σε Αλανίνη ώστε η θέση αυτή να μη φωσφορυλιώνεται in vivo, οδηγεί σε μείωση του ποσοστού των γεφυρών DNA με actin patches και αύξηση του ποσοστού των σπασμένων γεφυρών DNA. Αντίθετα, έκφραση της μεταλλαγμένης Src S51D, όπου η Σερίνη 51 έχει μεταλλαχθεί σε Ασπαραγινικό οξύ ώστε η θέση αυτή να μιμείται συνεχώς την φωσφορυλίωση, προστατεύει την δημιουργία των actin patches και εμποδίζει το σπάσιμο των γεφυρών DNA σε κύτταρα με μειωμένη έκφραση των Chk1 και Src πρωτεϊνών. Με βάση τα δεδομένα αυτά, προτείνουμε ένα νέο μηχανισμό σταθεροποίησης των γέφυρων DNA κατά την κυτταροκίνηση, μέσω διατήρησης των actin patches από τις πρωτείνες Chk1 και Src

Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM

Rapid Low Cost Vulnerability and Risk Assessment for Earthquake Planning: An Application to Rhodes City (Greece)

The geography and level of vulnerability and risk is an essential input to decision making for earthquake planning in urban areas. Nonetheless local agencies rarely have the capacity and resources to develop and maintain sophisticated GIS tools for this purpose. We argue that accuracy in risk and vulnerability assessment although desired, is not indispensable for earthquake planning at a local level. Rough assessments can be proved adequate provided that the delivered information is: a. structured in a manner that takes into account the local context, and b. comprehensive enough to encompass the range of probable earthquake ef-fects and emergency needs in the area. Should a tool to obtain such information is made available to the local authorities and if maintained in the long run, it can serve effectively decision making and planning. This could be the case even if it is used only for information manage-ment, demonstration exercises and training. Holding this point of view a method for a rapid low cost assessment of urban earthquake risk was developed based on open source statistical data from the National Statistical Survey of Greece. The method was applied to the capital city of Rhodes island (Greece) and the system was adjusted to the role of the Municipality of Rhodes in earthquake mitigation and emergency planning. It comprises the following steps: - An identification of the main categories of elements at risk in the city taking into account the development and urban profile of the area. - Development of thematic maps concerning the geography of these key elements of the urban environment, and especially land uses distribution, population geography, building densities, critical facilities, buildings of special use (such as schools and welfare institu-tions), tourism facilities. - Assessment of earthquake vulnerability of buildings taking into account statistical data on building features and based on general vulnerability curves elaborated by working groups of the Technical Chamber of Greece. - An estimation of expected losses related to the seismic hazard level in the area - A mainly qualitative assessment of urban earthquake risk and its distribution using com-binations of thematic information and event scenarios. Based on the above, a GIS tool was set up with the aim to facilitate earthquake awareness, decision making and planning at a local level. The tool was developed within the framework of the research project ESTIA that aimed at providing local authorities with advanced software and a comprehensive system to support decision making and risk management.JRC.G.7-Traceability and vulnerability assessmen

Emergency Management against Natural Hazards in the Acropolis of Athens

Using the case of the Acropolis of Athens, this paper aims to broaden current knowledge on risk and emergency management in archaeological complexes of high visitation. More specifically, it focuses on the protection of visitors and staff and intends to provide guidelines towards an emergency response plan for geodynamic and meteorological hazards in the Athens Acropolis archaeological site, along with a risk reduction and preparedness strategy. To this end, the paper first analyzes the main challenges arising from the everyday use of the archaeological site and the high visitor flows, mainly during summer. Secondly, it sets out the main parameters for drawing up an emergency evacuation plan for staff and visitors. Finally, it proposes preparedness guidelines, including training and information for all involved, together with a roadmap towards reducing existing risk and the implementation of necessary infrastructure interventions against residual risk. To finish, we conclude that challenges in emergency planning for the Acropolis of Athens do not arise solely from the unique conditions of the place and restrictions associated with the universal value of the monument but point towards reducing high residual risk and improving risk governance

Present and Future Research in Disaster Reduction Systems (7)

The objective of this paper is to summarize the research activities of the Research Center for Disaster Reduction Systems (DRS), DPRI. The activities include research seminars inviting guest speakers from various fields such as disaster risk manager of the public sector, researchers, education sector and mass-media. Camping involving academic staffs as well as students is also a highlight of our activities. Research projects funded by the government as well as outside funding bodies are introduced

巨大災害研究の現状と展望（7）

The objective of this paper is to summarize the research activities of the Research Center for Disaster Reduction Systems (DRS), DPRI. The activities include research seminars inviting guest speakers from various fields such as disaster risk manager of the public sector, researchers, education sector and mass-media. Camping involving academic staffs as well as students is also a highlight of our activities. Research projects funded by the government as well as outside funding bodies are introduced