The centrosome in cell cycle and
signal transduction pathways
Bodo M.H. Lange
Classically the centrosome is known as they provide a dominant site for micro- the elucidation of the centrosome com-
the microtubule organising centre in tubule nucleation and play a role in position (Andersen et al. 2003; Bornens
higher eukaryotic cells, coordinating positioning the spindle correctly in the 2002; Doxsey 2002; Wigge et al. 1998)
microtubule-dependent functions. Alt- cell (Rieder et al. 2001). however, we still don’t know the majori-
hough correct, this is only part of the Why does this cell organelle respond to ty of components of the centrosome. If
story of what a centrosome does. The global cellular signalling cues after all? we really want to untangle the molecu-
multitude of the microtubule-dependent One answer might be that it is part of a lar spider web of signal transduction, cell
functions in a cell (including intracellu- complex cellular protection mechanism cycle regulation and centrosome func-
lar transport, cell shape and polarity that brings the cell cycle to a stop when tion then, no doubt, we have to tackle
determination, motility, mitosis, cytoki- environmental conditions are harmful. this problem. Luckily, we have now new
nesis) demands that the microtubule At the moment we don’t know how such technologies and knowledge at our
organising centre is both regulated by signal transduction pathways are inter- hands and the next section will give an
and regulating cell cycle and signal connected to the centrosome and which overview on how we are addressing this
transduction events. In this sense the centrosomal proteins are substrates for problem in our laboratory.
centrosome is "more" than a microtubu- signal transducer molecules. For sure,
le-organising centre, playing an active some of the answers to these questions A systematic screen of centrosome
role also in cell cycle progression, stress lay in the molecular composition and composition and function
response and checkpoint control, via organisation of the centrosome and thus In our work we are focusing on three
interactions with numerous signal trans- reflecting its multiple functions. In this fundamental questions: (1) How is the
duction molecules (Lange 2002). For respect, good progress has been made in centrosome integrated in cell cycle and
example damaged or incompletely repli-
cated DNA triggers centrosome destruc-
tion in Drosophila early embryos (Takada
et al. 2003) suggesting an interconnec-
tion of the centrosome to DNA damage
signalling pathways. In addition, follo-
wing destruction of the centrosome in
cultured cells by laser beam irradiation,
a mitotic spindle is formed, chromoso-
mes are separating, but the cells under-
go abnormal cytokinesis and fail to exit
the G1 phase of the cell cycle (Khodjakov
& Rieder, 2001). This data suggests that
the centrosome has a primary role in
cytokinesis and cell cycle progression
rather than in spindle formation. Indeed,
there is not an absolute requirement for
cells to possess a centrosome to form
spindles. Spindles can be formed
without centrosomes in naturally occur-
ring systems such as in Drosophila oocy-
tes (Theurkauf & Hawley, 1992) or in
vitro systems (Heald et al. 1996) where
motor proteins rather than centrosomes
have been proposed to mediate spindle
formation. This does not mean, however,
Fig. 1: Immunofluoresence microscopy image of the preblastoderm stage of a Drosophila
that centrosomes are superfluous for embryo labeled with an antibody against phosphorylated histone H3. The condensing
spindle and microtubule organisation, prophase chromosomes, shown in red, demonstrate the synchronous duplication and
because when centrosomes are present division cycles in the early embryo syncytium.
18 Zellbiologie aktuell · 30. Jahrgang · Ausgabe 1/2004
In order to answer these questions we cost effective method to knock down
first need to have more complete infor- protein expression from a gene of known
mation of the molecular make-up of the sequence (Figure 3). The obtained phe-
centrosome. The centrosome has so far notypes are classified according to their
escaped detailed biochemical and func- relevance for centrosome function and
tional characterisation due to the small integration in signal transduction path-
quantities of protein obtained through ways. Subsequently, relevant proteins
organelle isolation methods and due to are cloned, their localisation is verified
lack of genetic approaches to study its through GFP-tagged expression and
function in higher eukaryotes. However, characterised in flies. Furthermore, with
new and improved methods, such as bet- the biochemical characterisation of the-
ter isolation techniques (Lange et al. se proteins we identify specific protein-
2000a; Müller, Lehmann & Lange unpu- protein interactions between novel or
blished), improved sensitivity of mass known centrosomal and signalling mole-
spectrometry (Yarmush & Jayaraman, cules. In summary, we developed a very
2002) and RNA interference (Clemens et efficient functional screen that integra-
al. 2000) are making a systematic bio-
chemical and functional dissection of
this organelle feasible.
To address these questions we initiated a
systematic analysis of the molecular
components of the centrosome using an
affinity-purification isolation method in
combination with mass spectrometry
protein identification. We are using
Drosophila as a model system for this
work. Why Drosophila? First, Drosophila
is an organism that is genetically mani-
pulable and its genome has been
sequenced. Therefore, we can study the
function of individual molecules in the
context of tissue and throughout deve-
lopment, providing functional clues that
would be absent in a cell culture system.
Secondly, the syncytial (cell membrane
free) early embryonic stage of Drosophi-
Fig. 2: (A, B) Immunofluoresence microscopy
images of isolated Drososphila centrosomes
la is well suited for the isolation of cen-
labelled with an anti- -tubulin antibody. trosomes (Figure 1): these embryos are
(A) Centrosomes isolated by sucrose density essentially tiny "bags”, not constrained
centrifugation. (B) Centrosomes further purified by cellular borders, full of cell organelles
by immuno-affinity isolation on magnetic beads and native protein complexes that are
coupled to antibodies. Beads are shown in red,
centrosomes in yellow. (C) MALDI mass spectro-
easily amenable to biochemical isola-
metry fingerprinting of the centrosomal prepara- tion. Thirdly, growing large fly popula-
tions isolated from coomassie-stained gels. The tions we can obtain per day 30-50 g of
most abundant band identified (green circle) is the embryo starting material, required for
protein CNN. The diagram shows the hits of the the systematic mass isolation and cha-
peptide mass distribution that was used for the
identification of the CNN protein, after tryptic
racterisation of centrosomes.
digest (work in collaboration with Gustavsson To isolate centrosomes we developed an
& Gobom). immuno-affinity approach using magne-
tic beads (Figure 2). This method has the
Fig. 3: Schematic flow diagram of the RNAi
advantage that most contaminants functional screen: Once centrosomal proteins
cell signalling pathways? (2) What holds (which otherwise co-migrate with cen- have been identified by MALDI mass spectrometry,
the structure of the centrosome together trosomes in biochemical fractionation dsRNA is generated by PCR amplification from
and constitutes the scaffold for the methods) are removed. In this way we genomic target DNA sequences. dsRNA is used for
assembly of functional protein comple- the protein “knock-down” assay in cultured Droso-
have identified over 60 centrosomal pro-
phila cells. Phenotypes are analysed by immuno-
xes onto the centrosome? (3) Which tein candidates. We are screening those fluorescence microscopy: centrosomes are labelled
molecular events mediate the change of candidate molecules for their relevance with anti- -tubulin antibodies (green), DNA is
microtubule nucleation capacity of the by RNAi in Drosophila cultured cells. stained with DAPI (blue) and microtubules with
centrosome during the cell cycle? This is a fast, extremely efficient and anti- -tubulin antibody (red).
Zellbiologie aktuell · 30. Jahrgang · Ausgabe 1/2004 19
tes the direct biochemical identification get kinases to Hsp90, it was likely that los, P. Becker, A. Hyman and E. Karsenti. 1996. Self-
of novel centrosomal proteins with their the observed phenotypes could be organization of microtubules into bipolar spindles
around artificial chromosomes in Xenopus egg
functional characterisation. The follo- brought about by the inactivation of one extracts. Nature 382, 420-425.
wing sections describe two examples of or more kinases. Indeed, the inactivation Khodjakov, A. and C.L. Rieder. 2001. Centrosomes
the application of this approach. of Hsp90 and Cdc37 lead to degradation enhance the fidelity of cytokinesis in vertebrates
of Aurora B kinase in mammalian cells and are required for cell cycle progression. J. Cell
Centrosome function depends on and in Drosophila (Lange et al. 2002). Biol. 153, 237-242.
Hsp90 Hence, the requirement of the Lange, B.M.H. 2002. Integration of the centrosome
One of the centrosomal proteins identi- Cdc37/Hsp90 complex for mitotic pro- in cell cycle control, stress response and signal
fied, heat shock protein 90 (Hsp90) is a transduction pathways. Curr. Opin. Cell Biol. 14,
cesses and microtubule organisation is 35-43.
molecular chaperon maintaining the evolutionary conserved.
Lange, B.M.H., E. Rebollo, A. Herold, and C. Gonza-
stability and activity of signalling mole-
lez. 2002. Cdc37 is essential for chromosome
cules (Lange et al. 2000a). Hsp90 is pre- Outlook segregation and cytokinesis in higher eukaryotes.
sent in the Drosophila centrosome in dif- Our approach integrates the direct bio- EMBO J., 21, 5364-5374.
ferent developmental and cell cycle sta- chemical identification of novel centro- Lange, B.M.H., A. Bachi, M. Wilm and C. Gonzalez.
ges, in the early embryo as well as in lar- somal proteins with their functional 2000a. Hsp90 is a core centrosomal component
val spermatocytes. Moreover, Hsp90 and is required at different stages of the centroso-
characterisation. This combination suc-
me cycle in Drosophila and vertebrates. EMBO J.
localisation at the centrosome is conser- cessfully exploits the advantages of the 19, 1252-1262.
ved in all vertebrate cells from chicken excellent biochemical and genetic sys- Lange, B.M.H., A.J. Faragher, P. March and K. Gull.
to human. We demonstrated that Hsp90 tem of Drosophila. So far, we have iden- 2000b. Centriole duplication and maturation in
is a core centrosomal protein necessary tified over 60 candidate centrosomal animal cells. Current Topics Dev. Biol. 49, 235-249.
for the maintenance of centrosome proteins which are currently being cha- Rieder, C.L., S. Faruki, and A. Khodjakov. 2001. The
function and integrity, required for the racterised. With this system at hand, the centrosome in vertebrates: more than a microtu-
fidelity of chromosome segregation and combination of cell biology approaches bule-organizing center. Trends Cell Biol. 11:413-
cell cycle progression (Lange et al. with the expertise and facilities for high-
2000a). Abrogation of Hsp90 function in Takada, S, A. Kelkar and W.E. Theurkauf. 2003. Dro-
throughput analysis at the Max-Planck sophila checkpoint kinase 2 couples centrosome
Drosophila or in mammalian cells results Institute for Molecular Genetics in Berlin function and spindle assembly to genomic integri-
in abnormal centrosomal and mitotic provides the opportunity to obtain a ty. Cell 11, 87-99.
phenotypes. In particular, centrosomal genome wide view of protein complex Theurkauf, W.E. and R.S. Hawley. 1992. Meiotic
separation and maturation (i.e. the structure and in vivo function. In addi- spindle assembly in Drosophila females: Behavior
increase in pericentriolar material during tion, the structural characterisation of of nonexchange chromosomes and the effects of
mutations in the nod kinesin-like protein. J. Cell
the cell cycle; Lange et al. 2000b) are centrosomal protein complexes is ongo- Biol. 116, 1167–1180.
affected, resulting in cells with fragmen- ing, as part of the Ultra-Structural Net-
Wigge, P.A., O.N. Jensen, S. Holmes, S. Soues, M.
ted and abnormal in size centrosomes. work (USN) in Berlin/Berlin-Branden- Mann and J.V. Kilmartin. 1998. Analysis of the Sac-
Furthermore, lack of Hsp90 function burg, which is aiming at the high- charomyces spindle pole by matrix-assisted laser
triggers abnormal spindle formation throughput isolation and structural ana- desorption/ionization (MALDI) mass spectrometry.
with the subsequent chromosome segre- J. Cell Biol. 141, 967-977.
lysis of protein complexes via cryo-elec-
gation resulting in a high percentage of tron microscopy. The elucidation of cen- Yarmush, M.L. and A. Jayaraman. 2002. Advances
aneuploid and polyploid cells. In sum- in proteomic technologies. Annu. Rev. Biomed. Eng.
trosome structure and function will help 4, 349-373.
mary, Hsp90 has an essential role in cen- us to understand the molecular events
trosome function and mitosis in higher and the regulation of cell division and
Anschrift des Verfassers:
eukaryotic cells. signal transduction in flies and humans.
PD Dr. Bodo M. H. Lange
Integration of microtubule organi- Max-Planck Institute for Molecular Genetics
References Department of Vertebrate Genomics
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Cdc37, the kinase-targeting unit of racterization of the human centrosome by protein Tel.: +49-30-8413-1645
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