Current Biology 17, 834–843, May 15, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.04.036
Article
Overexpressing Centriole-Replication
Proteins In Vivo Induces Centriole
Overduplication and De Novo Formation
Nina Peel,
1
Naomi R. Stevens,
1
Renata Basto,
1
and Jordan W. Raff
1,
*
1
The Gurdon Institute
Tennis Court Road
Cambridge CB2 1QN
United Kingdom
Summary
Background: Centrosomes have important roles in
many aspects of cell organization, and aberrations in
their number and function are associated with various
diseases, including cancer. Centrosomes consist of
a pair of centrioles surrounded by a pericentriolar matrix
(PCM), and their replication is tightly regulated. Here, we
investigate the effects of overexpressing the three
proteins known to be required for centriole replication
in Drosophila—DSas-6, DSas-4, and Sak.
Results: By directly observing centriole replication in
living Drosophila embryos, we show that the overex-
pression of GFP-DSas-6 can drive extra rounds of cen-
triole replication within a single cell cycle. Extra centri-
ole-like structures also accumulate in brain cells that
overexpress either GFP-DSas-6 or GFP-Sak, but not
DSas-4-GFP. No extra centrioles accumulate in sper-
matocytes that overexpress any of these three proteins.
Most remarkably, the overexpression of any one of
these three proteins results in the rapid de novo forma-
tion of many hundreds of centriole-like structures in un-
fertilized eggs, which normally do not contain centrioles.
Conclusions: Our data suggest that the levels of cen-
triolar DSas-6 determine the number of daughter centri-
oles formed during centriole replication. Overexpres-
sion of either DSas-6 or Sak can induce the formation
of extra centrioles in some tissues but not others, sug-
gesting that centriole replication is regulated differently
in different tissues. The finding that the overexpression
of DSas-4, DSas-6, or Sak can rapidly induce the de
novo formation of centriole-like structures in Drosophila
eggs suggests that this process results from the stabili-
zation of centriole-precursors that are normally present
in the egg.
Introduction
The centrosome is the main microtubule-organizing
center in animal cells, and it consists of a pair of centri-
oles surrounded by an amorphous pericentriolar matrix
(PCM) [1]. An increase in centrosome number is often
associated with cancer and may contribute to tumor
progression [2, 3]. Although centrosomes are dispens-
able for cell division in some systems [4, 5], extra cen-
trosomes can lead to multipolar mitotic spindles and
thereby to chromosomal instability, which is a character-
istic of many cancers. Centrioles are also required for
templating the growth of cilia—conserved structures
that have diverse and essential roles in development
[6]. It is critical, therefore, that each daughter cell inherits
a single centriole pair after mitosis.
Duplication of the centrioles is a highly ordered
process that is tightly coupled to the cell cycle, and it
usually occurs in close apposition to an existing cen-
triole [7, 8]. Genome-wide RNAi and genetic screens in
C. elegans have identified five proteins required for cen-
triole duplication—SPD-2, ZYG-1, SAS-5, SAS-6, and
SAS-4 [9–16]. These proteins localize to centrioles and
act in sequence to orchestrate centriole duplication in
the worm embryo. SPD-2 helps recruit ZYG-1 to the
centrioles and ZYG-1 then recruits SAS-5 and SAS-6
[17, 18]. The loss of SAS-5 or SAS-6 blocks formation
of the central tube of the daughter centriole, an early
event in the initiation of centriole duplication. SAS-4 is
the last protein recruited and it is required for the addi-
tion of singlet microtubules to the perimeter of the cen-
tral tube [18].
Several of the proteins involved in centriole duplica-
tion in C. elegans have been shown to have similar roles
in other species. Sak and Plk4 are Drosophila and
human protein kinases that are very weakly related to
ZYG-1, and they are both required for centriole duplica-
tion [19, 20]
. SAS-4 has orthologs in humans (CenpJ/
CPAP) and Drosophila (DSas-4), and DSas-4 is essential
for centriole duplication in flies [4]. The human ortholog
of SAS-6, HsSAS-6, is a centriolar protein that is essen-
tial for centriole duplication [10, 12]. The function of the
putative Drosophila ortholog of SAS-6 (CG15524) has
not been reported.
Importantly, the overexpression of human Plk4 or
HsSAS-6 results in the formation of extra centriole-like
structures in cultured cells [10, 20], which, in the case
of Plk4, have the ultrastructural appearance of centri-
oles [20]. Thus, the overexpression of at least some of
the centriole-duplication proteins is sufficient to drive
the formation of extra centrioles, although the mecha-
nism is unknown. It is also unknown whether over-
expression of these proteins in vivo can lead to extra
centrioles: the production of extra centrioles was not
reported when SAS-6 or HsSAS-6 were expressed in
C. elegans embryos [10, 12].
Although centriole formation usually occurs on a pre-
existing centriole that serves as a template, in some cir-
cumstances centrioles can form de novo. Such de novo
centriole formation is a normal part of the development
of some organisms, including the early mouse embryo,
parthenogenetic Hymenopteran insects, and the amoeba
Naegleria gruberi [21–23]. It can also be induced by the
ablation of the centrioles in cells that would normally rely
on templated centriole production [5, 24, 25]. It seems
that many cell types have the ability to form centrioles
de novo but that the presence of a single centriole sup-
presses this pathway [24, 26].
*Correspondence: j.raff@gurdon.cam.ac.uk