TY - JOUR A1 - Schulz, Irene A1 - Erle, Alexander A1 - Gräf, Ralph A1 - Krueger, Anne A1 - Lohmeier, Heiner A1 - Putzler, Sascha A1 - Samereier, Matthias A1 - Weidenthaler, Sebastian T1 - Identification and cell cycle-dependent localization of nine novel, genuine centrosomal components in Dictyostelium discoideum N2 - 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. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/36113/ U6 - https://doi.org/10.1002/Cm.20384 SN - 0886-1544 ER - TY - JOUR A1 - Schulz, Irene A1 - Baumann, Otto A1 - Samereier, Matthias A1 - Zoglmeier, Christine A1 - Gräf, Ralph T1 - Dictyostelium Sun1 is a dynamic membrane protein of both nuclear membranes and required for centrosomal association with clustered centromeres N2 - 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. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/01719335 U6 - https://doi.org/10.1016/j.ejcb.2009.06.003 SN - 0171-9335 ER - TY - JOUR A1 - Schulz, Irene A1 - Erle, Alexander A1 - Gräf, Ralph A1 - Krueger, Anne A1 - Putzler, Sascha A1 - Samereier, Matthias A1 - Weidenthaler, Sebastian T1 - Cell cycle-dependent localization of novel centrosomal and centromeric proteins in Dictyostelium Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/01719335 U6 - https://doi.org/10.1016/S0171-9335(09)00023-5 SN - 0171-9335 ER - TY - JOUR A1 - Samereier, Matthias A1 - Baumann, Otto A1 - Meyer, Irene A1 - Gräf, Ralph T1 - Analysis of dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of dictyostelium microtubules JF - Cellular and molecular life sciences N2 - 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. KW - Dictyostelium KW - TACC KW - DdCP224 KW - XMAP215 KW - Microtubules KW - Centrosome Y1 - 2011 U6 - https://doi.org/10.1007/s00018-010-0453-0 SN - 1420-682X VL - 68 IS - 2 SP - 275 EP - 287 PB - Springer CY - Basel ER -