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

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

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.

Blotting, Western, Cell Line, Centrioles/metabolism, Centrosome/metabolism, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins/metabolism, Microscopy, Microscopy, Fluorescence/methods, Microtubules/metabolism, Nerve Tissue Proteins/metabolism, RNA Interference
1465-7392
1159-1168
Mennella, V
43c60e29-c0a7-4ab8-8e5c-fcb59f70a28a
Keszthelyi, B
96d732be-4ccc-484c-ab7d-ec2edaf11ebd
McDonald, K L
b9eb9c32-6643-4e94-81fc-eb2ec3fa9b0b
Chhun, B
34cb449e-9874-4621-9047-b9d0e0bb9933
Kan, F
01387fa8-b014-40d1-94db-4b2221bd330f
Rogers, G C
6c6aa9e9-3500-4777-9046-a77cb0c463d1
Huang, B
8213448b-060c-4d70-b898-d00c6ac754a5
Agard, D A
c0733eec-3b98-4a05-9cac-4a965769f5b1
Mennella, V
43c60e29-c0a7-4ab8-8e5c-fcb59f70a28a
Keszthelyi, B
96d732be-4ccc-484c-ab7d-ec2edaf11ebd
McDonald, K L
b9eb9c32-6643-4e94-81fc-eb2ec3fa9b0b
Chhun, B
34cb449e-9874-4621-9047-b9d0e0bb9933
Kan, F
01387fa8-b014-40d1-94db-4b2221bd330f
Rogers, G C
6c6aa9e9-3500-4777-9046-a77cb0c463d1
Huang, B
8213448b-060c-4d70-b898-d00c6ac754a5
Agard, D A
c0733eec-3b98-4a05-9cac-4a965769f5b1

Mennella, V, Keszthelyi, B, McDonald, K L, Chhun, B, Kan, F, Rogers, G C, Huang, B and Agard, D A (2012) Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nature Cell Biology, 14 (11), 1159-1168. (doi:10.1038/ncb2597).

Record type: Article

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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More information

Published date: November 2012
Keywords: Blotting, Western, Cell Line, Centrioles/metabolism, Centrosome/metabolism, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins/metabolism, Microscopy, Microscopy, Fluorescence/methods, Microtubules/metabolism, Nerve Tissue Proteins/metabolism, RNA Interference

Identifiers

Local EPrints ID: 434038
URI: https://eprints.soton.ac.uk/id/eprint/434038
ISSN: 1465-7392
PURE UUID: 985304ea-1643-475e-a602-f5f29fd1e18d
ORCID for V Mennella: ORCID iD orcid.org/0000-0002-4842-9012

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Date deposited: 11 Sep 2019 16:30
Last modified: 03 Dec 2019 01:20

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Contributors

Author: V Mennella ORCID iD
Author: B Keszthelyi
Author: K L McDonald
Author: B Chhun
Author: F Kan
Author: G C Rogers
Author: B Huang
Author: D A Agard

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