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Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium

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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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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.

Author information

Correspondence to Ralph Gräf.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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

 

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

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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Keywords

  • Dictyostelium
  • Corona
  • Microtubules
  • Centrosome
  • Nucleus