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Nucleolus |
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The nucleolus is a subnuclear organelle
formed around the ribosomal DNA (rDNA) repeats, which
cluster at chromosomal loci called nucleolar organizing
regions (NORs). It is the factory in which 28S, 18S and
5.8S ribosomal RNAs (rRNAs) are transcribed, processed
and assembled into ribosomal subunits.
In addition to its major role in ribosome production,
the nucleolus may also be involved in messenger RNA (mRNA)
export or degradation. Recent studies suggest that the
mammalian nucleolus may also play roles in tumourigenesis,
viral replication and cellular stress sensoring. However,
the pathway and the identities of the molecular machineries
involved in these mechanisms within this nuclear organelle
remain largely unknown. |
SEM image of a mammalian nucleus,
with arrow pointing to nucleolus |
SEM image of purified nucleoli |
Due to its inherent high
density, nucleoli from cultured human cells can be isolated
in great quantities from sonicated nuclear extracts.
Taking advantage of this, our laboratory and others
have previously employed various mass spectrometry techniques
to identify the protein components from highly purified
nucleolar preparations. Visit the Protocols
page on the Lamond Lab website for a detailed nucleolar
isolation protocol, including still images and Quicktime
movies demonstrating key steps.
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Fluorescent protein-tagging
experiments and photobleaching analyses have demonstrated
the dynamic nature of the nucleolar proteome, where
proteins accumulate in the nucleolus only under specific
metabolic conditions or at specific cell cycle stages.
Recently, we have extended our mass spectrometry analyses
to measure the dynamic behaviour of the nucleolar proteome
by quantitating the relative level of individual nucleolar
components upon transcriptional inhibition using a method
known as Stable Isotope Labelling with Amino acids in
Cell Culture ( SILAC). |
DIC image of a nucleolus following
inhibition of transcription (green is overlay of GFP-PSP1,
which accumulates in "caps" at the periphery
of the nucleolus) |
For more information, check out our "Science at a Glance"
poster and article in the Journal of Cell Science:
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Lam, Y.W., Trinkle-Mulcahy,
L. and Lamond, A.I. The Nucleolus. J. Cell Sci., 118:1335-1337,
2005.
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