Abstract
The Dictyostelium 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.
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References
Azimzadeh J, Marshall WF (2010) Building the centriole. Curr Biol 20:R816–R825
Dictenberg JB, Zimmerman W, Sparks CA, Young A, Vidair C, Zheng YX, Carrington W, Fay FS, Doxsey SJ (1998) Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J Cell Biol 141:163–174
Fong KW, Choi YK, Rattner JB, Qi RZ (2008) CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the {gamma}-tubulin ring complex. Mol Biol Cell 19:115–125
Haren L, Stearns T, Lüders J (2009) Plk1-dependent recruitment of gamma-tubulin complexes to mitotic centrosomes involves multiple PCM components. PLoS One 4:e5976
Conduit PT, Brunk K, Dobbelaere J, Dix CI, Lucas EP, Raff JW (2010) Centrioles regulate centrosome size by controlling the rate of Cnn incorporation into the PCM. Curr Biol 20:2178–2186
Conduit PT, Raff JW (2010) Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts. Curr Biol 20:2187–2192
Gräf R, Daunderer C, Schulz I (2004) Molecular and functional analysis of the dictyostelium centrosome. Int Rev Cytol 241:155–202
Ding R, West RR, Morphew M, Oakley BR, McIntosh JR (1997) The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Mol Biol Cell 8:1461–1479
Ueda M, Schliwa M, Euteneuer U (1999) Unusual centrosome cycle in Dictyostelium: correlation of dynamic behavior and structural changes. Mol Biol Cell 10:151–160
Daunderer C, Gräf R (2002) Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex. Eur J Cell Biol 81:175–184
Daunderer C, Schliwa M, Gräf R (2001) Dictyostelium centrin-related protein (DdCrp), the most divergent member of the centrin family, possesses only two EF hands and dissociates from the centrosome during mitosis. Eur J Cell Biol 80:621–630
Rehberg M, Gräf R (2002) Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation. Mol Biol Cell 13:2301–2310
Rehberg M, Kleylein-Sohn J, Faix J, Ho TH, Schulz I, Gräf R (2005) Dictyostelium LIS1 is a centrosomal protein required for microtubule/cell cortex interactions, nucleus/centrosome linkage, and actin dynamics. Mol Biol Cell 16:2759–2771
Gräf R, Euteneuer U, Ho TH, Rehberg M (2003) Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number. Mol Biol Cell 14:4067–4074
Schulz I, Erle A, Gräf R, Kruger A, Lohmeier H, Putzler S, Samereier M, Weidenthaler S (2009) Identification and cell cycle-dependent localization of nine novel, genuine centrosomal components in Dictyostelium discoideum. Cell Motil Cytoskeleton 66:915–928
Blau-Wasser R, Euteneuer U, Xiong H, Gassen B, Schleicher M, Noegel AA (2009) CP250, a novel acidic coiled-coil protein of the Dictyostelium centrosome, affects growth, chemotaxis, and the nuclear envelope. Mol Biol Cell 20:4348–4361
Samereier M, Baumann O, Meyer I, Gräf R (2011) Analysis of Dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of Dictyostelium microtubules. Cell Mol Life Sci 68:275–287
Reinders Y, Schulz I, Gräf R, Sickmann A (2006) Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches. J Proteome Res 5:589–598
Gräf R, Euteneuer U, Ueda M, Schliwa M (1998) Isolation of nucleation-competent centrosomes from Dictyostelium discoideum. Eur J Cell Biol 76:167–175
Euteneuer U, Gräf R, Kube-Granderath E, Schliwa M (1998) Dictyostelium gamma-tubulin: molecular characterization and ultrastructural localization. J Cell Sci 111:405–412
Kaller M, Euteneuer U, Nellen W (2006) Differential effects of heterochromatin protein 1 isoforms on mitotic chromosome distribution and growth in Dictyostelium discoideum. Eukaryot Cell 5:530–543
Schulz I, Baumann O, Samereier M, Zoglmeier C, Gräf R (2009) Dictyostelium Sun1 is a dynamic membrane protein of both nuclear membranes and required for centrosomal association with clustered centromeres. Eur J Cell Biol 88:621–638
Xiong H, Rivero F, Euteneuer U, Mondal S, Mana-Capelli S, Larochelle D, Vogel A, Gassen B, Noegel AA (2008) Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability. Traffic 9:708–724
Samereier M, Meyer I, Koonce MP, Gräf R (2010) Live cell-imaging techniques for analyses of microtubules in Dictyostelium. Methods Cell Biol 97:341–357
Gräf R, Daunderer C, Schliwa M (2000) Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication. J Cell Sci 113:1747–1758
McIntosh JR (1985) Spindle structure and the mechanisms of chromosome movement. Basic Life Sci 36:197–229
Brito DA, Strauss J, Magidson V, Tikhonenko I, Khodjakov A, Koonce MP (2005) Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium. Mol Biol Cell 16:3334–3340
Yingling J, Youn YH, Darling D, Toyo-Oka K, Pramparo T, Hirotsune S, Wynshaw-Boris A (2008) Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division. Cell 132:474–486
Karsenti E, Vernos I (2001) The mitotic spindle: a self-made machine. Science 294:543–547
Zheng Y (2004) G protein control of microtubule assembly. Annu Rev Cell Dev Biol 20:867–894
Rieder CL, Faruki S, Khodjakov A (2001) The centrosome in vertebrates: more than a microtubule-organizing center. Trends Cell Biol 11:413–419
Kraemer N, Issa L, Hauck SC, Mani S, Ninnemann O, Kaindl AM (2011) What’s the hype about CDK5RAP2? Cell Mol Life Sci 68:1719–1736
Lucas EP, Raff JW (2007) Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin. J Cell Biol 178:725–732
Starr DA, Fridolfsson HN (2010). Interactions between nuclei and the cytoskeleton are mediated by SUN-KASH nuclear-envelope bridges. Annu Rev Cell Dev Biol
Ma S, Triviños Lagos L, Gräf R, Chisholm RL (1999) Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium. J Cell Biol 147:1261–1274
Kitanishi T, Shibaoka H, Fukui Y (1984) Disruption of microtubules and retardation of development of Dictyostelium with Ethyl N-phenylcarbamate and thiabendazole. Protoplasma 120:185–196
Martens H, Novotny J, Oberstrass J, Steck TL, Postlethwait P, Nellen W (2002) RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase. Mol Biol Cell 13:445–453
Fukui Y, Yumura S, Yumura TK (1987) Agar-overlay immunofluorescence: high resolution studies of cytoskeletal components and their changes during chemotaxis. Methods Cell Biol 28:347–356
Wehland J, Willingham MC (1983) A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements. J Cell Biol 97:1476–1490
Gräf R, Daunderer C, Schliwa M (1999) Cell cycle-dependent localization of monoclonal antibodies raised against isolated Dictyostelium centrosomes. Biol Cell 91:471–477
Acknowledgments
We would like to thank Belinda Pipke for technical assistance. We also acknowledge Dr. Annette Müller-Taubenberger for providing the mCherry-H2B plasmid and Dr. Alexandra Lepier for critically reading the manuscript. This work was supported by DFG GR1642/3-1 and GR1642/4-1.
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18_2011_904_MOESM2_ESM.mov
Supplementary material 2 (MOV 3287 kb). Movie S1. Mitosis in GFP-CP148/mCherry-H2B cells. GFP-CP148 is shown in green, histone-labeled chromosomes are shown in red. The mitotic cell is visible in the middle. Mitotic stages can be judged from the extent of DNA condensation and progression of chromosome segregation. GFP-CP148 disappears from the centrosome in prophase. In telophase, several GFP-CP148 foci appear in the cytosol and migrate in a radial fashion towards the nucleus where they join at the position of the new centrosome. Confocal spinning disk microscopy; time lapse acquisition rate was four stacks per minute (at a frame rate of 10 fr/s); maximum intensity projection of nine slices per image stack
Supplementary material 3 (MOV 1269 kb). Movie S2. GFP-CP148 shows almost no fluorescence recovery after photobleaching in GFP-CP148/cherry-H2B cells. GFP-CP148 is shown in green, histone-labeled chromosomes are shown in red. Left, centrosome bleached with a point-focused 473-nm laser pulse at time point 170 s; right, non-bleached control cell. Confocal spinning disk microscopy; time lapse acquisition rate was six stacks per minute (at a frame rate of 10 fr/s); maximum intensity projection of seven slices per image stack
18_2011_904_MOESM4_ESM.mov
Supplementary material 4 (MOV 6634 kb). Movie S3. Two mitoses in CP148 RNAi/GFP-α-tubulin cells (upper left, lower left). After duplication each mitotic MTOC sits at the pole of a bipolar spindle and organizes astral microtubules. During cytokinesis, the radial arrangement of astral microtubules becomes disrupted again. Confocal spinning disk microscopy; time lapse acquisition rate was four stacks per minute (at a frame rate of 10 fr/s); maximum intensity projection of eight slices per image stack
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Kuhnert, O., Baumann, O., Meyer, I. et al. Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium . Cell. Mol. Life Sci. 69, 1875–1888 (2012). https://doi.org/10.1007/s00018-011-0904-2
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DOI: https://doi.org/10.1007/s00018-011-0904-2