TY - JOUR A1 - Meyer, Irene A1 - Peter, Tatjana A1 - Batsios, Petros A1 - Kuhnert, Oliver A1 - Krueger-Genge, Anne A1 - Camurca, Carl A1 - Gräf, Ralph T1 - CP39, CP75 and CP91 are major structural components of the Dictyostelium JF - European journal of cell biology N2 - The acentriolar Dictyostelium centrosome is a nucleus-associated body consisting of a core structure with three plaque-like layers, which are surrounded by a microtubule-nucleating corona. The core duplicates once per cell cycle at the G2/M transition, whereby its central layer disappears and the two outer layers form the mitotic spindle poles. Through proteomic analysis of isolated centrosomes, we have identified CP39 and CP75, two essential components of the core structure. Both proteins can be assigned to the central core layer as their centrosomal presence is correlated to the disappearance and reappearance of the central core layer in the course of centrosome duplication. Both proteins contain domains with centrosome-binding activity in their N- and C-terminal halves, whereby the respective N-terminal half is required for cell cycle-dependent regulation. CP39 is capable of self-interaction and GFP-CP39 overexpression elicited supernumerary microtubule-organizing centers and pre-centrosomal cytosolic clusters. Underexpression stopped cell growth and reversed the MTOC amplification phenotype. In contrast, in case of CP75 underexpression of the protein by RNAi treatment elicited supernumerary MTOCs. In addition, CP75RNAi affects correct chromosome segregation and causes co-depletion of CP39 and CP91, another central core layer component. CP39 and CP75 interact with each other directly in a yeast two-hybrid assay. Furthermore, CP39, CP75 and CP91 mutually interact in a proximity-dependent biotin identification (BioID) assay. Our data indicate that these three proteins are all required for proper centrosome biogenesis and make up the major structural components of core structure's central layer. KW - Dictyostelium KW - Mitosis KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2017 U6 - https://doi.org/10.1016/j.eicb.2017.01.004 SN - 0171-9335 SN - 1618-1298 VL - 96 SP - 119 EP - 130 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Kuhnert, Oliver A1 - Baumann, Otto A1 - Meyer, Irene A1 - Gräf, Ralph T1 - CP55, a novel key component of centrosomal organization in dictyostelium JF - Cellular and molecular life sciences N2 - Dictyostelium centrosomes consist of a layered core structure surrounded by a microtubule-nucleating corona. At the G2/M transition, the corona dissociates and the core structure duplicates, yielding two spindle pole bodies. Finally, in telophase, the spindle poles mature into two new, complete centrosomes. CP55 was identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed that during interphase the majority of centrosomal GFP-CP55 is immobile, which indicates a structural task of CP55 at the centrosome. The CP55null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their partially disorganized Golgi apparatus. KW - Dictyostelium KW - Corona KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2012 U6 - https://doi.org/10.1007/s00018-012-1040-3 SN - 1420-682X VL - 69 IS - 21 SP - 3651 EP - 3664 PB - Springer CY - Basel ER - TY - JOUR A1 - Kuhnert, Oliver A1 - Baumann, Otto A1 - Meyer, Irene A1 - Gräf, Ralph T1 - Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium JF - Cellular and molecular life sciences N2 - The centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates, yielding two spindle poles. CP148 is a novel coiled coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle-dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage. KW - Dictyostelium KW - Corona KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2012 U6 - https://doi.org/10.1007/s00018-011-0904-2 SN - 1420-682X VL - 69 IS - 11 SP - 1875 EP - 1888 PB - Springer CY - Basel ER -