Article

Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation

Molecular Cancer Biology Program and Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland.
Molecular & Cellular Proteomics (Impact Factor: 6.56). 07/2011; 10(10):M111.009241. DOI: 10.1074/mcp.M111.009241
Source: PubMed

ABSTRACT

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

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Available from: François-Michel Boisvert, Mar 11, 2014
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    • "Changes in temperature[27,48], ionic strength[47,48], and chemotoxic and DNA damage[27,59,60,78,79]are environmental changes known to disrupt phase separated cellular bodies and in vitro liquid droplets. The stiffness of nucleoli isolated from HeLa cells was decreased or increased upon RNA Polymerase or proteasome inhibition, respectively, based on atomic force microscopy measurements[79]. "
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    • "There is wide variation in nucleolar response to stress and DNA damage, although it is generally considered the hub for directing the stress response [27]. For example, in response to cellular UV exposure, the nucleolus segments into nucleolar caps [55]. In our studies, the nucleolus was sensitive to even low levels of dnTRF2 expression as early as 12 hours after induction, as exhibited by nucleolar fragmentation and necklace formation. "
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