Review
R487
Centrioles take center stage
Wallace F. Marshall
Centrioles are among the most beautiful and
mysterious of all cell organelles. Although the
ultrastructure of centrioles has been studied in great
detail ever since the advent of electron microscopy,
these studies raised as many questions as they
answered, and for a long time both the function and
mode of duplication of centrioles remained
controversial. It is now clear that centrioles play an
important role in cell division, although cells have
backup mechanisms for dividing if centrioles are
missing. The recent identification of proteins
comprising the different ultrastructural features of
centrioles has proven that these are not just figments of
the imagination but distinct components of a large and
complex protein machine. Finally, genetic and
biochemical studies have begun to identify the signals
that regulate centriole duplication and coordinate the
centriole cycle with the cell cycle.
Address: Department of Molecular, Cellular, and Developmental
Biology, Yale University, New Haven, Connecticut 06520, USA.
E-mail: [email protected]
Current Biology 2001, 11:R487–R496
0960-9822/01/$ – see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Centrioles are cylindrical structures found at the center of
the centrosome. Centrioles act as seeds to recruit microtubule nucleating material — referred to as pericentriolar
material — to give rise to a centrosome. Centrioles also act
as structural templates to initiate the assembly of cilia and
flagella, and in this role they are referred to as basal
bodies. Centrioles were an object of intense research in
the early days of cell biology, because of their central
location within the cell division machinery and their apparent self-replication. Early observations raised three central
questions of centriole biology that have remained unanswered to this day: what is the function of centrioles in cell
division; what is the molecular structure of centrioles; and
how do centrioles duplicate?
Recent data on the role of centrioles in centrosome assembly, cytokinesis and cell-cycle progression have sparked
a resurgence of interest in these questions. Clearly, a
genetic approach to centrioles will be essential. Unfortunately, yeast cells do not contain centrioles, but instead
have morphologically distinct structures called spindle pole
bodies. Despite tremendous advances in understanding
spindle pole bodies, these studies have not yet answered
the main questions of centriole biology, simply because it
is unclear how spindle pole bodies are related to centrioles. Only two homologs of spindle pole body proteins
have been found in animal genomes: centrin, the homolog
of Cdc31, and kendrin, the homolog of Spc110 [1]. Until
we identify more components of centrioles, we will lack
the molecular Rosetta stone that would let us apply our
knowledge of yeast spindle pole bodies to animal centrioles. Consequently, genetic approaches to centrioles have
instead turned to genetic model organisms that have centrioles similar to those of mammalian cells, including Drosophila, Caenorhabditis elegans, and the yeast-like green alga
Chlamydomonas.
In this review, I shall summarize the current state of our
biochemical, genetic and ultrastructural understanding of
centrioles, and how this information is leading to answers
to the central questions about the function, composition
and duplication of centrioles.
Centriole function
The primary function of centrioles during cell division is to
recruit microtubule nucleating factors into a discrete focus,
the centrosome (Figure 1). Animal cells lacking centrioles
do not form centrosomes, as judged by the accumulation of
pericentriolar material into discrete microtubule organizing
centers [2–5]. When centrioles are dissolved by injection of
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Current Biology Vol 11 No 12
Figure 1
Normal centriole number
Two centriole pairs
Two centrosomes
Astral bipolar spindle
Proper mitotic segregation
Not enough centrioles
One centriole pair
One centrosome
Monastral bipolar spindle
Chromosome loss
No centrioles
No centrosomes
Anastral bipolar spindle
Chromosome loss
Too many centrioles
Multiple centriole pairs
Multiple centrosomes
Multipolar spindle
Chromosome loss
Current Biology
Centriole function in cell division. Centrioles nucleate assembly of
centrosomes by recruiting microtubule nucleating material into a
discrete focus in the cell; centrioles (green), chromosomes (blue),
microtubule nucleating pericentriolar material (red), microtubules (black
lines). Cells with too few centrioles form either monopolar or monastral
bipolar spindles if just one centriole pair is present, and form anastral
bipolar spindles if no centrioles are present. These abnormal spindles
cannot be properly oriented during cytokinesis, leading to errors in
cytokinesis and chromosome segregation. Cells with too many
centrioles form multipolar spindles, again leading to chromosome
segregation errors. Errors in centriole duplication thus lead to
chromosome loss and genomic instability.
antibodies recognizing a centriole-specific tubulin isoform,
the centrosomes disperse, and only re-assemble when the
centrioles are restored [4], demonstrating a requirement for
centrioles in centrosome assembly and maintenance.
Cells lacking centrioles can still form bipolar spindles
[5–8]. This was once thought to imply that centrioles are
not needed to make centrosomes, but it turns out that
bipolar spindles in centriole-less cells form via an alternative, non-centrosomal pathway not normally used in somatic
cells [5]. In this case, microtubules nucleated by the chromosomes self-organize into a bipolar spindle with the aid
of motor proteins [9,10]. This alternative spindle assembly
pathway is common in meiosis [3], consistent with the lack
of both centrioles and centrosomes in meiotic cells. Thus,
cells lacking centrioles do not form centrosomes, but can
assemble spindles using this alternate pathway. The fact
that cells without centrioles cannot form centrosomes,
which are required to make astral microtubules, explains
a long-standing observation that acentriolar spindles are
always anastral [3,5,11–13]. Because astral microtubules
are thought to position the spindle during cytokinesis, we
would predict that cells lacking centrioles would show
defects in spindle orientation and cytokinesis. This is
indeed the case.
One piece of evidence linking centrioles with cytokinesis
came from the bld2 mutation in the unicellular green alga
Chlamydomonas. Mutant bld2 cells have severe defects in
centriole structure, so that instead of normal centrioles,
bld2 cells only contain thin rings of singlet microtubules
[14]. Careful analysis of cell division in bld2 mutant strains
revealed that, while spindle assembly and mitosis proceeded without any problems, the spindle was often misoriented with respect to the cleavage furrow, resulting in
cytokinesis errors [15]. These relatively minor defects in
cell division were found with a partial loss-of-function
allele of bld2. In contrast, a null allele of bld2 is lethal [16],
suggesting that perhaps the structurally defective centriole
formed by the original bld2 mutant cells retains some
essential function. As discussed below, centrioles are now
thought to contribute directly to cell-cycle progression,
and this may explain the lethality in bld2 null mutants.
Consistent with the results in algae, the removal or
ablation of centrioles from mammalian cells also leads to
errors in cytokinesis [17–19]. These errors probably result
from an inability of the resulting anastral spindles to maintain the correct position as the cleavage furrow progresses.
It has long been known that, if mitotic spindles are displaced within a cell during cytokinesis, they can induce
new cleavage-furrow formation in the new position
[20–22]. Failure of cytokinesis in acentriolar cells may
result from an inability to anchor the spindle in one place
long enough to establish or maintain a single cleavage
furrow. Moreover, completion of cleavage appears to coincide with, and may be triggered by, the close approach and
subsequent departure of the mother centriole from the
midbody [18], implying that centrioles may have a second,
more direct role in regulating cytokinesis.
Further evidence that centriole-nucleated centrosomes position spindles relative to the cleavage furrow in cytokinesis
Review Centrioles
has recently been obtained through analysis of mutations
in two Drosophila genes, asterless (asl) and centrosomin (cnn).
In cells mutant for either of these genes, a functional centrosome fails to assemble around the centriole, as judged
by accumulation of centrosomal proteins such as γ-tubulin
and CP190. As a result, such cells display a marked reduction of astral microtubules at spindle poles [23,24]. Importantly, homozygous asl [25] mutant embryos, or cnn mutant
embryos from homozygous cnn mutant female flies (which
will completely lack centrosomin protein), fail to develop
and show dramatic defects in cell division [24,26], confirming that centrosomes, and by extension centrioles, are essential for proper animal development. The importance of
centrioles in animal development has been further supported by the finding that mutations in ZYG-1, a centrosome-associated kinase that is required for centriole duplication in C. elegans, results in a complete failure of embryonic development [27].
The defects in development that are caused by centriole
and centrosome mutations are probably not due to a simple
failure in spindle assembly, because bipolar spindles can
assemble without centrosomes. Examination of weak
mutants of asl that survive through larval development
showed that spindle orientation was defective during the
asymmetric neuroblast divisions, consistent with a role for
astral microtubules in spindle orientation [28]. In homozygous cnn mutant offspring of heterozygous mothers, the
maternal contribution of Cnn protein is sufficient to allow
development to adulthood. However, detailed analysis of
cell division in these mutant flies revealed a reduction in
astral microtubules that was accompanied by a significant
defect in asymmetric neuroblast division [26], similar to
the results with asl mutants. These results thus confirm
the importance of centriole-nucleated centrosomes in orienting the spindle during cytokinesis, presumably via the
astral microtubules.
In addition to playing a role in cell division via nucleating
astral microtubules, centriole-nucleated centrosomes have
recently been found to play a role in regulating cell-cycle
progression. Cells from which centrioles have been
removed, either by microsurgery or laser ablation, progress
through mitosis but arrest in G1 of the following cell cycle
and never reach S phase [17,19]. These studies imply that
centrioles can directly influence cell-cycle progression. This
may reflect the existence of a checkpoint that monitors centriole copy number and arrests cell division in cells with
too few centrioles, thus avoiding chromosome segregation
errors in subsequent cell divisions.
Clearly, centrioles play an important role in cell division,
both by forming the centrosome, thus giving rise to astral
spindle poles that can be properly positioned relative to the
cleavage furrow, and also by directly influencing cell-cycle
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progression, possibly by recruiting cell-cycle regulatory molecules onto centrioles. Yet neither of these functions is
strictly essential for cell division. Instead, it appears that
centrioles have evolved as a way of ensuring high fidelity
of chromosome segregation and spindle placement [29], by
imposing spatial regulation on the inherently self-organizing mitotic apparatus.
Centriole structure and composition
A major limiting factor in investigating centriole function
is our current ignorance of the molecular composition of
centrioles. A more detailed knowledge of centriole molecular architecture would allow genetic and reverse genetic
techniques to be brought to bear on investigations of
centriole function.
Centriole core structure
Each centriole consists of a nine-fold symmetrical array of
triplet microtubules, called blades (Figure 2). The distal
end contains the plus-ends of the microtubules, and templates the assembly of cilia and flagella when centrioles turn
into basal bodies. The other, proximal end of the centriole
contains the ‘cartwheel’, a set of nine spokes connected to
a central axis. The proximal end is also the site of new centriole assembly. While a significant number of components
of the microtubule triplets have been identified, mainly as
a byproduct of studies on flagellar doublet microtubules,
much less is known about what molecules might compose
the interior of the centriole.
The microtubule triplet blades contain α and β tubulin.
Specific post-translational modifications of tubulin occur
in the centriole blades. In particular, polyglutamylated
tubulin is clearly very important for maintenance of centriole integrity [4]. Additional protein components of the
triplet blades include Sp77, Sp83, Rib43 and tektin [30–32],
proteins of unknown function that were first identified as
structural components of flagellar microtubule doublets.
These data suggest that a common set of underlying molecular interactions establish the parallel microtubule doublets
and triplets seen in centrioles and cilia.
Nothing whatsoever is known about the molecular composition of the cartwheel structure. γ-tubulin is located within
the lumen of the centriole barrel, near the proximal end
where the cartwheel is found [33]. Other proteins located
at the proximal end of the centriole include the NIMA
(never in mitosis A)-related kinase Nek2 and the Nek2interacting coiled-coil protein C-Nap1 [34]. (The founding
member of the NIMA family is a protein kinase that controls initiation of mitosis in Aspergillus nidulans.) Overexpression of Nek2 [35], as well as interference with C-Nap1
function either by antibody blocking or by expressing
dominant-negative constructs [36], causes centrosomes to
split, suggesting that C-Nap1, under control of Nek2,
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Figure 2
Distal lumen
Vfl1
Centrin
Appendages
p210
Cenexin?
p52
Centriole structure. Schematic diagram
showing main ultrastructural features of
centrioles, indicating known protein
constituents. The distal appendages function
in membrane anchoring when the centriole
acts as a basal body and migrates to the
plasma membrane. The satellites are a focus
of microtubule nucleation during interphase,
but not mitosis. The proximal end of centriole
is site of nucleation of new centrioles during
centriole duplication, as well as the region of
attachment for several types of centrioleassociated fibers.
Satellites
PCM-1
Kendrin?
Blades
αβ-tubulin
δ-tubulin
Sp77
Sp83
Rib43
Tektins
Fibers
sf-assemblin
Centrin
BAp95
BAp90
Cartwheel
Composition unknown
Proximal lumen
γ-tubulin
Nek2
C-Nap1
Current Biology
maintains the linkage between the proximal ends of the
two centrioles.
At the other end of the centriole, the distal lumen of the
barrel contains the ‘EF-hand’ protein centrin [37,38] and
the coiled-coil protein Vfl1p, which localizes to the inner
wall of the distal lumen [39]. Other proteins located at the
distal end include the microtubule-severing protein katanin
[40] and Fa1p, a protein involved in regulating detachment
of flagella from basal bodies [41]. Interestingly, another
protein involved in flagellar detachment, Fa2p, has been
found to be a homolog of Nek2 (L. Quarmby, personal
communication), suggesting that NIMA family kinases may
play multiple roles in centriole structure and function.
One other putative centriole component needs to be
mentioned, namely DNA. Based on a variety of biochemical, histochemical, and genetic experiments, it was once
thought that centrioles might contain their own DNAbased genomes, but subsequent work has absolutely ruled
out this possibility [42].
Centriole accessory structures
Centrioles bristle with more attached superstructures
than a World War II battleship. These include fibers,
protruding appendages and fuzzy balls. Although these
structures were all seen by electron microscopy over
30 years ago, their molecular composition is only now
being determined.
Review Centrioles
A set of nine short appendages extend from the distal end
of the centriole [43]. When a centriole becomes a basal
body, these distal appendages — also known as transition
fibers — connect the centriole with the plasma membrane. Studies in algae have identified one distal appendage
protein, p210, which shares homology with the clathrin
coat assembly protein AP180 [44], consistent with a role
in attachment to membranes. Electron micrographs have
shown association of membrane vesicles with the distal
end of centrioles in the process of becoming basal bodies
[45], possibly via an interaction with the distal appendages.
Another likely component of the appendages is cenexin,
which localizes to the same general region as the distal
appendages [46] and is only found on the mother centriole, which is normally the only one to contain appendages
in mammalian cells [43]. (During centriole duplication,
the pre-existing centriole is called the mother centriole,
and the new centriole that forms next to it is called its
daughter.)
Fuzzy balls called satellites are found within the pericentriolar material, and appear to be attached to the centriole
barrel by striated stems [43]. The function of satellites is
unclear, although it has been observed that, in interphase
but not mitotic cells, most of the microtubules emanating
from the centrosome have their minus ends embedded in
the satellites [43]. So far only one protein, PCM-1, has been
localized to the satellite [47]. Consistent with a role for the
satellite in attaching cytoplasmic microtubules during
interphase, PCM-1 can interact with microtubules via cytoplasmic dynein [47]. PCM-1 is present on centrosomes
during G1, but dissociates from centrosomes during G2 and
M phases [48], exactly when the satellites stop acting as
microtubule organizing sites. PCM-1 has recently been
found in a complex with kendrin [49], a coiled coil protein
homologous to the yeast spindle-pole-body protein Spc110
[1]. Spc110 is involved in recruiting microtubule nucleating material to the nuclear face of the spindle pole body,
suggesting that its homolog kendrin might be involved in
microtubule nucleation by the satellites.
The two centrioles present in the cell in interphase are
joined by various connecting fibers. In algae there are two
fibers, the distal connecting fiber and the proximal connecting fiber. The distal connecting fiber contains the
protein centrin, while the proximal connection contains
the protein BAp90 [50]. These fibers fail to assemble in
vfl1 and vfl3 mutants of Chlamydomonas; consequently,
centriole segregation does not occur properly, leading to
variable numbers of centrioles per cell [51,52]. Evidently
these fibers keep the centriole pair together until it is time
to separate them in a controlled fashion. Based on protein
localization and mutant phenotypes, it is likely that Vfl1p
and C-Nap1 are required to anchor the distal and proximal
connecting fibers, respectively, to the centrioles.
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A variety of different fibers extend outward from the
proximal end of the centriole into the rest of the cell.
These fibers have mainly been observed and studied in
ciliates and algae, but are present in mammalian cells
[43]. Studies in green algae have revealed the protein
components of three distinct types of centriole-associated
fiber: the striated microtubule-associated fibers, composed of the segmented coiled-coil protein SF-assemblin
[53]; the ominously named sinister fibers, composed of a
different segmented coiled-coil protein BAp95 [54]; and
the nucleus–basal body connector fibers, composed of the
EF-hand protein centrin [55–57]. Another structure associated with the proximal end of the centriole is a fibrous ring
of material aptly named the ‘halo’ [58,59], the function and
composition of which are completely unknown.
Centriole duplication
The complex structure of the centriole raises the question,
how are new centrioles assembled? Centrioles undergo a
highly precise duplication, such that each centriole gives rise
to exactly one new centriole per cell cycle. Indeed, centrioles are the only cellular structures besides chromosomes to
undergo such a precise discrete duplication. This duplication process is necessary to maintain the exact number of
centrioles per cell; there is now strong evidence that abnormalities in centriole number accompany, and probably contribute directly to, tumor progression [60–64].
Centrioles duplicate by a remarkable process whose mechanism is completely unknown. New centrioles form adjacent to, and at right angles to, pre-existing centrioles. The
daughter centriole does not incorporate any part of the
mother centriole [65] and hence is not simply generated
by a splitting process. Why new centrioles form only next
to old ones is one of the longest-running questions of centriole biology. The most obvious model is that centrioles
contain an essential template structure needed to produce
a new centriole, so that new centrioles simply cannot form
except when nucleated by a pre-existing centriole. But
de novo assembly of centrioles has been demonstrated by
careful microscopic studies on parthenogenetic development of unfertilized oocytes [66,67], suggesting that preexisting centrioles are not strictly needed to make new
centrioles. Other examples of de novo centriole assembly
have been seen in certain lower plants [68], oomycetes
[69] and protists [70,71].
If centrioles can form de novo, then why does centriole
assembly normally start only next to pre-existing centrioles? Prions provide a possible analogy: prions normally are
switched to the infective conformation by pre-existing prion
proteins, but under certain conditions they can attain the
altered conformation spontaneously, albeit at a very low
frequency. In this model, spontaneous de novo centriole
assembly would normally be a very slow process that could
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not compete with normal templated assembly. However,
under certain circumstances, such as in oocytes loaded
with high concentrations of centriole precursor proteins,
mass action might drive centriole assembly to occur spontaneously even without pre-existing centrioles present.
This hypothesis is supported by the fact that the number
of centrioles formed de novo in artificially activated sea
urchin eggs is strongly dependent on the strength of activation [72]. After the activation is ended, further centriole
assembly occurs by the templated mechanism, suggesting
that once the driving stimulus is removed, de novo assembly reverts to being an inefficient process that cannot
compete with templated assembly. These data suggest
that de novo assembly might be a unique feature of certain
cell types that are developmentally primed to generate
large numbers of centrioles. Further evidence that de novo
assembly might only occur in specialized cells containing
massive stockpiles of precursor proteins has come from
experiments in which centrioles were removed from somatic
cells. The resulting centriole-less cells never recovered
centrioles [73,74], supporting the idea that de novo assembly might simply be too slow in somatic cells to compete
with templated assembly.
Experiments measuring the rate of de novo centriole
assembly in Chlamydomonas cells, however, have argued
against this model. Mutants with defects in centriole segregation frequently produce centriole-less progeny cells.
In contrast to mammalian cells, which arrest in G1 after
centrioles are removed [17,19], centriole-less Chlamydomonas cells continue to divide, and centrioles re-form
de novo at a rate only about two-fold slower than templated
assembly [75]. This indicates that de novo assembly is fast
enough to compete with templated assembly, and therefore that pre-existing centrioles, in addition to catalyzing
new centriole assembly in their immediate vicinity, also
must negatively regulate de novo assembly elsewhere in
the cell [75].
Experiments with cell-cycle arrest mutants showed that
de novo centriole assembly occurs in S phase of the cell
cycle and cannot occur in G1-arrested cells [75]. This fact
explains why de novo centriole assembly was not seen in
the earlier experiments on mammalian cell fragments that
are missing centrioles [73]. Because centriole-less mammalian cells arrest in G1 [17,19], they never reach the
correct cell-cycle stage for de novo assembly to occur. The
lack of de novo assembly in such these cells reflects the
cell-cycle-specific regulation of centriole assembly, rather
than the inherent ability of centrioles to form de novo. Thus
we conclude that new centrioles normally form next to old
ones, not because they cannot form elsewhere, but because
this de novo assembly pathway is normally down-regulated
under the influence of pre-existing centrioles.
In certain cells, de novo assembly appears to be exploited
as a way of rapidly generating large numbers of centrioles.
During early embryogenesis in the wasp Nasonia, centrioles
form de novo all around the cortex of the embryo, even in
fertilized embryos that contain centrioles [76]. Evidently,
in this case the suppression of de novo centriole assembly
by pre-existing centrioles is not functioning. Another
example is ciliogenesis in mammalian ciliated epithelia,
when centrioles form de novo in clusters that are spatially
separated from the original centrioles inherited in the previous division [45], again suggesting that de novo assembly
can occur in cells that contain centrioles.
Coordinating the centriole cycle with the cell
cycle
Centriole duplication is restricted not just spatially, through
the influence of pre-existing centrioles, but also temporally,
under the control of the cell-cycle engine. The complex
structure of the centriole assembles in a series of discrete
steps (Figure 3), each occurring at a different stage of the
cell cycle [43,58,59,77–80]. In this section, we will discuss
the centriole duplication cycle, and its coordination with the
nuclear division cycle, by breaking the process down into
several steps based on morphological data.
Step 1: Initiation of daughter centriole assembly
Assembly of new centrioles begins when cells enter S
phase [78,79]. S phase is permissive for centriole duplication, and multiple rounds of centriole duplication can occur
in S-phase-arrested cells [81]. The initiation of centriole
duplication during S phase requires the activity of cyclindependent kinase 2 (Cdk2)–cyclin E in Xenopus eggs and
early embryos [82,83] and Cdk2–cyclin A in somatic cells
[84], and recent work has identified nucleophosmin as the
downstream target of Cdk2–cyclin E in this process [85].
The step driven by Cdk2–cyclin E or A is probably the key
control point in coordinating centriole duplication with the
cell cycle. Recently a novel centrosome-associated kinase,
ZYG-1, has been identified through a genetic screen in C.
elegans. Zyg-1 mutants have a complete failure in centriole
duplication, suggesting the ZYG-1 kinase plays a role in
triggering centriole duplication [27].
Which part of the centriole assembles first? A structure
called the generative disc has been reported as the first
morphologically recognizable step in centriole assembly in
Paramecium [86], and is a likely candidate for the precursor
of centriole assembly. Identifying the molecular components of the generative disc is thus a high priority for
understanding centriole duplication. The centriole satellites have also been proposed to initiate centriole assembly
[58]. However, satellites persist separately from the daughter centriole after centriole duplication is complete [59].
Moreover, only the mother centriole of a pair has an associated satellite structure, yet both centrioles give rise to new
Review Centrioles
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Figure 3
Centriole duplication cycle; centrioles (green),
chromosomes (blue), and pericentriolar
material (red), much of which is sequestered
in the nucleus during interphase. Numbers
refer to steps of centriole duplication as listed
in the text. (1) Initiation of centriole duplication.
(2) Formation of a disc-like pro-centriole of
nine singlet microtubules. (3) Conversion of
singlet microtubules into triplets.
(4) Elongation of procentriole into full-length
centriole. (5) Recruitment of mitotic
centromatrix and microtubule nucleating
material onto centrioles to produce
centrosomes. (6) Separation of centriole pairs
to form the two poles of a bipolar spindle.
(7) Detachment of daughter centriole from its
mother, causing loss of perpendicular
arrangement. (8) Disassembly of mitotic
centrosome. (9) Interconversion between
centriole and basal body.
HFH4
Uni1
Vfl1,3
Cdk2–cyclinE/A
Nucleophosmin
Centrin
Vfl3
1
9
γ-tubulin
2
8
Bld2
δ-tubulin
3
S
G1
G2
Telophase
The
centriole
cycle
4
Cdc25string
Anaphase
M
Metaphase
Prophase
7
Cdc20
fizzy
5
Pericentrin
γ-TuRC
NuMA
TPX2
CP60,190
Ran
Asp
Cnn
6
Centrin
Aurora ARK1 family kinase
Nek2
C-Nap1
Current Biology
daughter centrioles prior to division [77], suggesting the
satellite is not needed to initiate centriole assembly. We
conclude that the generative disc, and not the satellite, is
the most likely precursor to centriole assembly.
The molecules that are required for initiating centriole
duplication remain unknown. In yeast, the centrin homolog
Cdc31 forms the half-bridge structure that appears to give
rise directly to a new spindle pole body, suggesting that
centrin might play a similar role in centriole duplication
[87]. Indeed, inhibition of centrin function has been shown
to cause defects in both templated [88,75] and de novo [89]
centriole duplication. Other proteins involved in centriole
duplication are Vfl3p [75] and γ and η tubulin [90,91],
although it is difficult to distinguish between a block at
this stage versus a block at the singlet microtubule assembly
stage (step 2).
Step 2: Formation of a ring of nine singlet microtubules
The most obvious sign that centriole assembly has begun is
the formation of a ring of nine singlet microtubules called
the procentriole [77]. How is a large nine-fold symmetric
structure built? One way would be by packing interactions
between large subunits, similar to the mechanism by which
viral capsids attain their various symmetry forms. Alternatively, some small nine-fold rotationally symmetric structure,
possibly even just a single molecule, might self-assemble
and then propagate its symmetry to the entire structure.
Certain repeating sequences within an α helix can generate
a nine-fold symmetric arrangement of amino acids around
the circumference of the helix [92]. Spoke structures, such as
those seen in the cartwheel at the proximal end of the centrioles, could bind to such a sequence motif and extend out to
the perimeter of the generative disc, directing assembly of
microtubule singlets in the correct positions.
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Once the singlets form, additional microtubules assemble
alongside them, forming a ring of nine triplet microtubules. Generally, the microtubules form first doublets,
and then triplets [86]. In Chlamydomonas bld2 mutants,
centriole development arrests at the beginning of this stage,
because bld2 mutant centrioles contain only singlet microtubules. The Chlamydomonas mutant uni3, defective in the
gene for δ tubulin, allows doublets to form, but prevents
them from assembling into triplets [93].
Drosophila, the detachment of the daughter centriole from
the mother requires the activity of Cdc20fizzy, which
promotes exit from mitosis by activating the anaphase
promoting complex [80]. In Xenopus, proteasome inhibitors,
as well as antibodies blocking SCF ubiquitin ligase subunits — named after their main components, Skp1, Cullin
and an F-box protein — prevent G1 centriole detachment
[98]. These results imply that detachment involves ubiquitin-dependent proteolysis, perhaps to sever a physical
linkage between the mother and daughter centrioles.
Step 4: Elongation
Step 8: Disassembly of mitotic centrosome
After the triplet microtubules have initiated, the entire
structure elongates into the full-length centriole. Elongation
normally takes place during mitosis [59], and experiments
in Drosophila have shown that the phosphatase Cdc25String,
which regulates entry into mitosis, is required for centriole
elongation [80].
After mitosis, pericentriolar proteins are re-sequestered in
the newly forming nuclei. Centrosome disassembly is
accompanied by dramatic changes in centriole ultrastructure. For example, the electron-dense halo seen around
the mother centriole of each pair during mitosis disappears
after anaphase [43,78].
Step 5: Recruitment of the mitotic centrosome
Step 9 (optional): Conversion into basal bodies
When mitosis begins, proteins are recruited around the
centrioles to give rise to the mitotic centrosome. Many
centrosomal proteins, such as NuMA [94], are sequestered
in the nucleus during interphase, which may explain the
results of elegant experiments in which eggs of the ribbonworm Cerebratulus were microsurgically cut to produce
enucleated cell fragments. These fragments could be
induced to assemble microtubule asters containing de novo
assembled centrioles, but only if the microsurgery was performed after nuclear envelope breakdown [95]. Most of
the centrosome proteins do not interact with centrioles
directly, but are instead recruited by a fibrous matrix, the
‘centromatrix’, which assembles onto the centrioles [96].
During G1 phase, centrioles can become basal bodies to
form cilia and flagella. Basal body assembly requires formation of the transition region to nucleate flagellar assembly, and attachment to the cell surface. In mam-malian
cells, conversion of centrioles into basal bodies appears to
be under control of the forkhead transcription factor
HFH-4 [99].
Step 3: Assembly of microtubule triplet blades
Step 6: Separation of centriole pairs
Prior to mitosis, the centrosome must be split into two centrosomes in order to form two spindle poles. Splitting is
accompanied by a separation of the two mother–daughter
centriole pairs from each other. This separation appears to
be triggered by phosphorylation of centrin during the
G2–M transition [97], suggesting that centrin-based connecting fibers may keep the original centriole pair connected after each member of the pair has formed its own
daughter centriole. Centriole pair separation can also be
triggered by the kinase Nek2 [35], at least one target of
which is the protein C-Nap1 located at the base of the centriole. It is important to distinguish this pre-mitotic splitting event, in which pairs of centrioles separate from each
other, from the post-mitotic detachment (step 7), in which
the two centrioles within one pair separate from each other.
Step 7: Detachment of the centriole pair
As cells exit mitosis and enter G1 phase, the mother and
daughter centrioles detach from each other, losing
their mutually perpendicular arrangement [43,78,79]. In
Conclusions: deciphering the enigma
Centrioles are suddenly becoming less enigmatic. We are
now equipped with the molecular and genetic tools to take
studies of centriole biology beyond the limits of ultrastructural description. Genetic screens in Chlamydomonas,
Drosophila and Caenorhabditis elegans are currently fleshing
out the genetic pathway of centriole assembly. At the
same time, modern proteomic approaches are beginning to
catalog the parts list of all centriolar proteins [100], and
cell-free extract systems have been developed in which
centriole assembly and centrosome recruitment can be
reconstituted [101]. After having endured a century-long
bear market, centrioles are finally coming into their own.
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