@article{SamereierBaumannMeyeretal.2011,
  author    = {Samereier, Matthias and Baumann, Otto and Meyer, Irene and Gr{\"a}f, Ralph},
  title     = {Analysis of dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of dictyostelium microtubules},
  series = {Cellular and molecular life sciences},
  volume    = {68},
  journal   = {Cellular and molecular life sciences},
  number    = {2},
  publisher = {Springer},
  address   = {Basel},
  issn      = {1420-682X},
  doi       = {10.1007/s00018-010-0453-0},
  pages     = {275 -- 287},
  year      = {2011},
  abstract  = {We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-alpha-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.},
  language  = {en}
}
@article{SchulzErleGraefetal.2009,
  author    = {Schulz, Irene and Erle, Alexander and Gr{\"a}f, Ralph and Krueger, Anne and Putzler, Sascha and Samereier, Matthias and Weidenthaler, Sebastian},
  title     = {Cell cycle-dependent localization of novel centrosomal and centromeric proteins in Dictyostelium},
  issn      = {0171-9335},
  doi       = {10.1016/S0171-9335(09)00023-5},
  year      = {2009},
  language  = {en}
}
@article{SchulzErleGraefetal.2009,
  author    = {Schulz, Irene and Erle, Alexander and Gr{\"a}f, Ralph and Krueger, Anne and Lohmeier, Heiner and Putzler, Sascha and Samereier, Matthias and Weidenthaler, Sebastian},
  title     = {Identification and cell cycle-dependent localization of nine novel, genuine centrosomal components in Dictyostelium discoideum},
  issn      = {0886-1544},
  doi       = {10.1002/Cm.20384},
  year      = {2009},
  abstract  = {The centrosome is the main microtubule-organizing center and constitutes the largest protein complex in a eukaryotic cell. The Dictyostelium centrosome is an established model for acentriolar centrosomes and it consists of a layered core structure Surrounded by a so-called corona, which harbors microtubule nucleation complexes. We have identified 34 new centrosomal candidate proteins through mass spectrometrical analysis of the proteome of isolated Dictyostelium centrosomes. Here we present a characterization of 12 centrosomal candidate proteins all featuring coiled coil regions and low expression levels, which are the most common attributes of centrosomal proteins. We used GFP fusion proteins to localize the candidate proteins in whole cells and on microtubule-free, isolated centrosomes. Thus we were able to identify nine new genuine centrosomal proteins including a putative orthologue of Cep192, an interaction partner of polo-like kinase 4 in human centriole biogenesis. In this respect, centrosomal localization of the only polo-like kinase in Dictyostelium, Pik, is also shown in this work. Using confocal deconvolution microscopy, four components, CP39, CP55, CP75, and CP91 could be clearly assigned to the so far almost uncharacterized centrosomal core structure, while CP148 and Cep192 localized to a zone between that of corona marker and core proteins. Finally, CP103 and CP248 were constituents of the corona. In contrast, NE81 was localized at the nuclear envelope and three others, an orthologue of the spindle checkpoint component Mad1, the novel Cenp68, and the centrosomal CP248 were observed at the centromeres, which are clustered and linked to the centrosome throughout the entire cell cycle. Cell Motil. Cytoskeleton 66: 915-928, 2009.},
  language  = {en}
}
@article{SchulzBaumannSamereieretal.2009,
  author    = {Schulz, Irene and Baumann, Otto and Samereier, Matthias and Zoglmeier, Christine and Gr{\"a}f, Ralph},
  title     = {Dictyostelium Sun1 is a dynamic membrane protein of both nuclear membranes and required for centrosomal association with clustered centromeres},
  issn      = {0171-9335},
  doi       = {10.1016/j.ejcb.2009.06.003},
  year      = {2009},
  abstract  = {Centrosomal attachment to nuclei is crucial for proper mitosis and nuclear positioning in various organisms, and generally involves Sun-family proteins located at the inner nuclear envelope. There is still no common scheme for the outer nuclear membrane proteins interacting with Sun I in centrosome/nucleus attachment. Here we propose a model in which Sun1 mediates a physical link between centrosomes and clustered centromeres through both nuclear membranes in Dictyostelium. For the first time we provide a detailed microscopic analysis of the centrosomal and nuclear envelope localization of endogenous Dictyostelium Sun1 during interphase and mitosis. By immunogold electron microscopy we show that Sun1 is a resident of both nuclear membranes. Disruption of Sun1 function by overexpression of full-length GFP-Sun1 or a GFP-Sun-domain deletion construct revealed not only the established function in centrosome/nucleus attachment and maintenance of ploidy, but also a requirement of Sun1 for the association of the centromere cluster with the centrosome. Live-cell imaging visualized the occurrence of mitotic defects, and demonstrated the requirement of microtubules for dynamic distance changes between centrosomes and nuclei. FRAP analysis revealed at least two populations of Sun1, with an immobile fraction associated with the centrosome, and a mobile fraction in the nuclear envelope.},
  language  = {en}
}
@phdthesis{Samereier2011,
  author    = {Samereier, Matthias},
  title     = {Functional analyses of microtubule and centrosome-associated proteins in Dictyostelium discoideum},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-52835},
  school      = {Universit{\"a}t Potsdam},
  year      = {2011},
  abstract  = {Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium.},
  language  = {en}
}