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Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization

Nature Cell Biology volume 14, pages 1159–1168 (2012)Cite this article

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Abstract

As the main microtubule-organizing centre in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. Although the importance of the PCM is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM subdiffraction-resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two main structural domains: a layer juxtaposed to the centriole wall, and proteins extending farther away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (PLP) reveals that its carboxy terminus is positioned at the centriole wall, it radiates outwards into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNA-mediated interference (RNAi), we show that PLP fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.

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Figure 1: 3D SIM of proteins critical for centrosome maturation identifies two distinct structural domains within the PCM.
Figure 2: The molecular architecture of PLP.
Figure 3: PLP fibrils associated with mother centrioles form a gap where the daughter centriole assembles.
Figure 4: PLP is associated exclusively with mother centrioles until metaphase.
Figure 5: Plp is required for the initial recruitment and proper 3D assembly of the PCM distal layer.
Figure 6: The molecular architecture of Kendrin/Pericentrin is similar to that of PLP.
Figure 7: Pericentrin-like protein forms elongated fibrils that extend radially from the centriole wall to support the 3D organization of the PCM.

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Acknowledgements

We especially thank J. Sedat, L. Winoto and C. Weisiger for invaluable assistance with the OMX, S. Li for advice and help with the 3D alignment strategy, and D. Buster and K. Model for comments and editing on the manuscript. We also would like to thank T. Avidor-reiss (Harvard University, USA), J. Raff (University of Oxford, UK), M. Bettencourt-Dias (Instituto Gulbenkian de Ciencia, Portugal), M. Gatti (Universitá La Sapienza di Roma, Italy), T. Megraw (Florida State University, USA), M. Takahashi (Teikyo Heisei University, Japan), Y. Zheng (Carnegie Institution of Washington) and Howard Hughes Medical Institute, T. Davis (University of Washington) and T. Kaufman (University of Indiana, USA) for generously sharing their antibodies. This work was financially supported by HHMI and NIH grant GM310627. B.H. is a recipient of the Searle Scholarship and the Packard Fellowship for Science and Engineering.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics and the Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94158, USA

    V. Mennella, B. Keszthelyi, F. Kan & D. A. Agard

  2. Electron Microscope Laboratory, University of California, Berkeley, California 94720, USA

    K. L. McDonald

  3. Department of Pharmaceutical Chemistry and Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94158, USA

    B. Chhun & B. Huang

  4. Department of Cellular and Molecular Medicine, Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724, USA

    G. C. Rogers

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Contributions

V.M. conceived the strategy, designed and performed experiments, analysed data and wrote the paper; B.K. and V.M., with input from others at UCSF, developed the 3D volume alignment and analysis procedure; K.L.M. performed the immuno-electron microscopy experiments; B.C. helped with STORM data acquisition; F.K. helped with immunostaining experiments; G.C.R. provided reagents and constructs; B.H. advised on data analysis and STORM experiments and discussed results; D.A.A. advised on data analysis, discussed results and wrote the paper.

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Mennella, V., Keszthelyi, B., McDonald, K. et al. Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat Cell Biol 14, 1159–1168 (2012). https://doi.org/10.1038/ncb2597

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