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Analysis of Dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of Dictyostelium microtubules

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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-α-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.

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Abbreviations

FRAP:

Fluorescence recovery after photobleaching

MAP:

Microtubule-associated protein

TBZ:

Thiabendazole

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Acknowledgments

We would like to thank Dr. Katrin Pfütze for her engagement in the early stages of this project. We also acknowledge Anita Guhlan and Anne Krumbiegel for technical assistance. We are grateful to Dr. Annette Müller-Taubenberger (University of Munich, Institute for Cell Biology, Munich, Germany) for the marsRFP plasmid. This work was supported by the Deutsche Forschungsgemeinschaft GR1642/2-2.

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Authors and Affiliations

  1. Department of Cell Biology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 26, 14476, Potsdam-Golm, Germany

    Matthias Samereier, Irene Meyer & Ralph Gräf

  2. Department of Animal Physiology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 26, 14476, Potsdam-Golm, Germany

    Otto Baumann

Authors
  1. Matthias Samereier
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  2. Otto Baumann
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  3. Irene Meyer
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  4. Ralph Gräf
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Corresponding author

Correspondence to Ralph Gräf.

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Fig. S1

Centrosomal dynamics of GFP-α-tubulin under the influence of the microtubule depolymerizing drug thiabendazole.A representative experiment of fluorescence recovery after photobleaching of centrosomal GFP-α-tubulin after incubation with 100 µM TBZ for 3 h is shown (the bleached region of interest was placed around the centrosome followed by 4D confocal live cell imaging. Fluorescence intensities in the bleached region of interest were evaluated with ImageJ. The half time of recovery and is indicated on the lower right (TIFF 14702 kb)

Fig. S2

Dynamic behavior of individual microtubules in the cell periphery of cells expressing marsRFP-α-tubulin and GFP-TACCdom.Two still images of movie 5 are shown. Tips of individual microtubules where movements are traceable are labeled with arrowheads (TIFF 23623 kb)

Movie 1

For FRAP analysis of centrosomal GFP-CP224 during interphase the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of 5 images with an interval of 0,5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 721 kb)

Movie 2

For FRAP analysis of centrosomal GFP-CP224 in TACC depleted cells during interphase the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of 5 images with an interval of 0,5 μm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 481 kb)

Movie 3

For FRAP analysis of spindle pole GFP-CP224 during mitosis the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 512 kb)

Movie 4

For FRAP analysis of spindle pole GFP-CP224 in TACC depleted cells during mitosis the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of 5 images with an interval of 0,5 μm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 1926 kb)

Movie 5

To study microtubule plus-end dynamics in vivo, a time series of confocal images of cells expressing marsRFP-α-tubulin and GFP-TACCdom as a microtubule plus-end marker were taken during interphase. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left(MPG 6434 kb)

Movie 6

For FRAP analysis of GFP-α-tubulin at microtubule plus ends the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 2816 kb)

Movie 7

For FRAP analysis of centrosomal GFP-α-tubulin during interphase the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 5248 kb)

Movie 8

For FRAP analysis of centrosomal GFP-α-tubulin in TACC depleted cells during interphase the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 4994 kb)

Movie 9

For FRAP analysis of spindle pole GFP-α-tubulin during mitosis the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 2492 kb)

Movie 10

For FRAP analysis of spindle pole GFP-α-tubulin in TACC depleted cells during mitosis the framed area was bleached followed by 4D confocal live cell imaging. For each time point, a z-stack consisting of five images with an interval of 0.5 µm was taken. Shown is a maximum intensity projection. The time interval is indicated on the upper left (MPG 3454 kb)

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Samereier, M., Baumann, O., Meyer, I. et al. Analysis of Dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of Dictyostelium microtubules. Cell. Mol. Life Sci. 68, 275–287 (2011). https://doi.org/10.1007/s00018-010-0453-0

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