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One to only two: a short history
of the centrosome and its duplication
Greenfield Sluder
rstb.royalsocietypublishing.org
Review
Cite this article: Sluder G. 2014 One to only
two: a short history of the centrosome and its
duplication. Phil. Trans. R. Soc. B 369:
20130455.
http://dx.doi.org/10.1098/rstb.2013.0455
One contribution of 18 to a Theme Issue
‘The centrosome renaissance’.
Subject Areas:
cellular biology
Keywords:
centriole, centrosome, duplication, template
Author for correspondence:
Greenfield Sluder
e-mail: [email protected]
Department of Cell and Developmental Biology, University of Massachusetts Medical School,
Worcester, MA 01655, USA
This review discusses some of the history of the fundamental, but not fully
solved problem of how the centrosome duplicates from one to only two as
the cell prepares for mitosis. We start with some of the early descriptions
of the centrosome and the remarkably prescient but then controversial inferences drawn concerning its function in the cell. For more than 100 years, one
of the most difficult issues for the concept of the centrosome has been to integrate observations that centrosomes appear to be important for spindle
assembly in animal cells yet are not evident in higher plant cells and some
animal cells. This stirred debate over the existence of centrosomes and
their importance. A parallel debate concerned the role of the centrioles in
organizing centrosomes. The relatively recent elucidation of bipolar spindle
assembly around chromatin allows a re-examination of the role of centrioles
in controlling centrosome duplication in animal cells. The problem of how
centrosomes precisely double in preparation for mitosis in animal cells has
now moved to the mystery of how only one procentriole is assembled at
each mother centriole.
1. Introduction
The centrosome, as the cell’s primary microtubule organizing centre (MTOC),
nucleates the interphase microtubule array that is central to many cellular processes [1]. In preparation for cell division, the centrosome duplicates, and in
mitosis, the sister centrosomes act in a dominant manner to determine the
essential bipolarity of the spindle. Because the purpose of mitosis is to divide
a mother cell into two genetically identical daughter cells, the cell must
ensure that the centrosome inherited from the previous mitosis doubles once
and only once. The presence of more than two centrosomes at mitosis greatly
increases the chances that the cell will distribute chromosomes unequally and
that some chromosomes may be profoundly damaged [2–4]. Genomic instability
can contribute to multi-step carcinogenesis (reviewed in [5–9]).
2. Early days
The first drawings of the centrosome are found in an 1876 study of mitosis in a
metazoan parasite of cephalopods by Van Beneden (reviewed in [10]). The centrosomes, labelled polar corpuscles, were drawn as dark dots or small filled
circles at each spindle pole. Little attention was given to these structures at
the time, but in 1883 and 1887, Van Beneden published studies on fertilization
and early cleavage stages of eggs of Ascaris megalocephala, a parasitic nematode
worm of horses and pigs. In these studies, he detailed a differentiated sphere of
cytoplasm at spindle poles from which astral and spindle fibres emanated. The
centre of each contained a more densely staining dot (see fig. 1.2 in [10]). Notably, he drew the doubling and separation of the dark dots during anaphase
and telophase. He proposed that what he now called the central corpuscles
were permanent self-replicating organelles of the cell.
Shortly thereafter, Theodor Boveri published two similar characterizations
of early Ascaris development in 1887 and 1888 (reviewed in [10,11]) describing
specializations at spindle poles he called centrosomes (see fig. 1.3 of [10] for
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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3. The ‘quadripartition’ experiment
A singularly important step forward in our understanding of
centrosome duplication was an elegantly thought out study
of the functional properties of centrosome duplication in
early-stage sea urchin zygotes [18]. At the time, nothing was
known about how centrosome duplication was controlled
and few, if any, were thinking about this issue.
In the late 1950s, Mazia was pursuing his hypothesis that
disulfide bonding was involved in the assembly of proteins
that formed spindle and astral fibres. His group found that
introduction of relatively high concentrations of –SH (they
used 0.075 M mercaptoethanol) rapidly disassembled spindles
in mitotic sand dollar and sea urchin zygotes [19]. They noted
the odd finding that the zygotes divided directly from one to
four after removal of the mercaptoethanol.
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showed, after fixation, staining and sectioning, aster-like
arrays of fibrillae and spindle-like fibre arrays between them
(reviewed in [11]). For the sceptics of the centrosome, the
dark dots of the centrioles could be written off as non-specific
cytoplasmic granules, and the fibre arrays could simply be
the action of the fixative on soluble cellular proteins. This
controversy over the reality of the centrosome was eventually
put to rest with the advent of electron microscopy in the
1950s that provided the first descriptions of centriole and centrosome ultrastructure (see [17] for a discussion of the interplay
between concepts of the centrosome and the technologies used
to define them).
Although the following story is not directly related to centrosomes, it illustrates the extent to which concerns over
fixation artefacts producing astral and spindle fibres had
become ingrained in the minds of cell biologists. In the
1950s at the Marine Biological Laboratory in Woods Hole,
Shinya Inoue showed an audience his polarization microscopy movie of a lilly pollen mother cell dividing. The
dynamic movements of the continuous spindle fibres and
the shortening of chromosomal fibre bundles in anaphase
were clearly evident. Given the nature of contrast generation
in the polarization microscope, this was unequivocal proof
for the existence of spindle fibres in vivo. At the end of the
talk, an accomplished investigator in the audience asked ‘Was
that cell alive?’ (S. Inoue 2005, personal communication).
Controversies of that time aside, by 1925, the concept of
the centrosome in animal cells could be reviewed in terms
that are remarkably contemporary today. Wilson [11] defined
the centrosome as ‘the central body lying at the astral center,
and constituting an autonomous cell-organ; the divisioncenter or dynamic center of the cell’. It was ‘the central
body (centrosome, centriole) about which as a center arise the
asters that form a conspicuous feature of many forms of mitotic cell-division. . .’. ‘The central body possesses in many cases
the power of growth and division and retains its morphological identity during interkinesis. . .’. ‘Its most constant and
essential component is the centriole, a minute granule or
rod, often double. . .commonly surrounded by the centrosome, lying at the center of the aster. It is often regarded as
an autonomous cell-organ arising only by the growth and
division of a pre-existing centriole.’ The question of how
the centrosome doubled from one to only two would, however, have to wait until the 1960 publication of a prescient
study from the laboratory of Daniel Mazia.
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photographs of dividing Ascaris zygotes taken by this author
from Boveri’s own slide preparations!). Boveri also proposed
the term centriole for the single or double dense staining dots
in the centre of these polar specializations. In time, centrosomes
were described in interphase, and dividing cells from a wide
variety of animal cells, though variations in morphology led
to the interchangeable use of the terms centriole and centrosome. Wilson [11] admitted to the difficulty of distinguishing
between the centriole and the centrosome and suggested the
more non-committal term ‘central body’.
These earliest reports were remarkable for the inferences
drawn about centrosome function from the collection of
dark staining dots, fibres and clear areas near the nucleus or
at spindle poles. Van Beneden [12] wrote ‘Thus we are justified
in regarding the attraction-sphere with its central corpuscle as
forming a permanent organ. . .of all cells; that every central
corpuscle is derived from a pre-existing corpuscle, every
attraction-sphere from the pre-existing sphere, and that division of the sphere precedes that of the cell-nucleus’. In the
same vein, Boveri said ‘the centrosome is an independent permanent cell-organ, which, exactly like the chromatic elements
[chromosomes] is transmitted by division to the daughtercells. (Quotes from Wilson [13].) Boveri concluded that the
central body can be regarded as the ‘dynamic centre’ of the
cell that coordinates nuclear and cytoplasmic division.
Boveri also appreciated the importance of centrosome copy
number. Polyspermic fertilization of echinoderm eggs led to
extra centrosomes which resulted in multipolar spindles and
improper distribution of chromosomes. In a prescient report
on the cytology of multipolar divisions in cancer cells, he proposed that abnormal numbers of centrosomes could change
the genomic content of daughter cells (Boveri [14]). Today,
consequences of extra centrosomes for genomic stability are
widely appreciated.
Unfortunately, Theodor Boveri died in 1915 at the relatively early age of 53 after repetitive bouts of fevers and
colics. Although the exact natures of his illnesses were not
fully known, they may have included an Ascaris worm infestation coming from the human pathogenic form that can also
inhabit pigs. Indeed, he confided to a friend shortly before his
death that ‘it is mean when the beasts you have worked on,
now start working on you’ (quoted from Maderspacher [15]).
Given the scientific mind, proposals concerning the existence, autonomy, permanence and importance of centrosomes/
centrioles soon became controversial (reviewed in [16,17]).
Centrosomes could not be seen throughout the cell cycle in
some cell types, and centrosomes/centrioles could not be
found in interphase or dividing higher plant cells despite careful investigation. Further scepticism about the permanence
and continuity of centrosomes arose from the pioneering
work of T.H. Morgan and Jacques Loeb in the late 1800s
which revealed that centrosomes could be experimentally
induced to assemble in unfertilized sea urchin eggs without
any apparent pre-existing structures—a phenomenon now
called de novo assembly. This suggested that centrosomes
were not permanent autonomous organs of the cell. Additional
doubts arose from claims that centrosomes and the fibres
emanating from them were simply fixation artefacts. Some
investigators demonstrated that artificial coagulation of
homogeneous protein solutions could lead to the formation
of alveolar formations such as those seen in fixed cells, as
well as astral rays and spindle-like structures. Even dead and
empty pith cells impregnated with an albumose solutions
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(a)
(b)
(c)
(d )
(h)
(i)
(j)
(k)
(g)
(l)
make only one spindle pole in the next cell cycle. The reproductive capacity of the centrosome was quantized; it had a
‘valence’ which could be experimentally manipulated. They
proposed that functionally each spindle pole contained two
linked mitotic centres that could split apart and separate
during prolonged mitosis to give a tetrapolar division. Each
mitotic centre was capable of elaborating a functional spindle
pole. After the four-way division, they proposed that each
single inherited mitotic centre duplicates to produce one
spindle pole of normal ‘valence’ with two linked mitotic
centres—hence a monopolar spindle. In effect, the splitting
and duplication of mitotic centres could be temporally
uncoupled under their experimental conditions. They proposed
that the reproductive cycle of the mitotic centres involved three
distinct and separable events: splitting, separation and duplication. Their timing experiments indicated that normally
these three events occur at the same time in telophase, a time
when DNA synthesis occurs in these rapidly cycling zygotes.
They concluded that centrosome duplication is not a fission process whereby the centrosome subdivides into sister bodies that
are equivalent to the original centrosome—a duplicative event
is needed.
Of course mercaptoethanol is a blunt instrument with many
effects. Subsequent investigation revealed that the relevant activity of mercaptoethanol in this context is to prolong
prometaphase long enough to allow the spindle poles to split.
Exactly the same phenomena, namely centrosome splitting
during prometaphase and assembly of monopolar spindles at
second mitosis, were observed in sea urchin zygotes when prometaphase was prolonged by colcemid at microtubule-specific
doses or by microsurgically cutting the first division spindle
into two half spindles thereby maintaining the activation of
the mitotic checkpoint [21]. Importantly, continuous observation of zygotes released from mecaptoethanol (or colcemid,
Phil. Trans. R. Soc. B 369: 20130455
Figure 1. (a – g) Diagrammatic representation of the experimental manipulation of the reproductive capacity of spindle poles in sea urchin zygotes. (a) The first
division spindle has a pair of centrioles at each spindle pole. (b,c) When prometaphase is prolonged, the centriole pairs with associated pericentriolar material split
without duplicating. The four spindle poles, each containing a single centriole, separate and a tetrapolar spindle is formed. (d ) In telophase, when DNA synthesis
begins in these zygotes, the single centrioles duplicate. (e) At second mitosis monopolar spindles are assembled, and each centrosome has the normal complement
of two centrioles. ( f,g) If two centrosomes of the tetrapolar spindle fail to completely separate (lower cell), a single nucleus is re-formed in one blastomere. At
mitosis a bipolar spindle is assembled; its poles have a normal complement of centrioles and consequently reproduce in a normal fashion in subsequent cell cycles.
(h – l ) Mitosis in blastomeres containing a monopolar spindle (enlarged diagram of the continued development of one of the cells shown in diagram (e). (h) Early in
second mitosis this blastomere contains a monopolar spindle. (i,j ) Prometaphase is often substantially longer than normal due to unattached kinetochores. The
spindle pole splits and the spindle reorganizes into a bipolar configuration with one centriole in each centrosome. (k) In telophase, each daughter cell inherits a
single centriole which duplicates. (l ) At third mitosis, monopolar spindles are again assembled in each daughter cell. The centrosome can split during prolonged
prometaphase yielding a bipolar spindle with a single centriole in each centrosome (not shown). Reproduced with permission from [20].
Further investigation of this intriguing four-way division
led to some surprising observations [18]. They treated sea
urchin zygotes coming into first mitosis with mercaptoethanol to disassemble the mitotic apparatus and hold the cells
in mitosis for roughly the duration of a cell cycle. Upon
release from the chemical, each cell immediately assembled
a tetrapolar mitotic apparatus (figure 1) and later divided
into four blastomeres. During prolonged metaphase, the
two spindle poles had split into four functional poles, and
after mitosis the four blastomeres each inherited a split
pole. In the second cell cycle, each of the four daughter
cells assembled a monopolar spindle. Blastomeres inheriting
split poles later divided in a bipolar manner suggesting that
the inherited split pole had functionally duplicated. If a cleavage furrow failed in a three-way division after release from
mercaptoethanol, then two monopolar spindles could come
together to form a functional bipolar spindle that supported
normal division.
The remarkable aspect of this study was the involved but
elegant interpretation of the results. At the outset, Mazia and
his co-workers were deliberate in saying that they were using
the behaviour of spindle poles to analyse the duplication of
‘mitotic centres’ not morphological structures such as the centrioles, though they were well aware that there might be
parallels. In concept, the mitotic centre was not the spindle
pole itself, but the seed or organizer around which the pole
(centrosome) was elaborated. Thus, the duplication and separation of the spindle poles reflected the duplication of
the mitotic centres. Although not said at the time, this
abstraction—the mitotic centre—freed them from becoming
enmeshed in any uncertainties over the role of centrioles in
spindle poles.
The key finding was that a spindle pole could split and do
so only once. In addition, a cell inheriting a split pole could
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(f)
(e)
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Starting in the late 1800s and continuing almost up to the
present, one of the most nettlesome issues for the concept
of centrosomes determining the spindle poles had been to
explain why higher plant cells and some animal cells can
assemble bipolar spindles without centrioles or evident centrosomes. Neither light nor electron microscopy revealed
recognizable specialized structures of the spindle poles of
these forms. Mazia [29] said ‘We may as well face the fact
that microscopy has not yet confirmed any general portrait
of the centrosomes as mitotic poles. . .’.
Mazia [18,24] was careful to separate the concept of the
mitotic centre (an activity) from the centriole (a morphological
structure in animal cells). Although he did not say at the time
5. Centrioles versus centrosomes
In the 1960s and early 1970s, the role of centrioles in organizing
centrosome/spindle poles in animal cells was debated. Some
felt that, when present, the centrioles themselves were the organizing principle for the centrosome (see [37,38]; reviewed
in [16]). Centrioles were at the right location and their duplication/separation tracked the behaviour of sister centrosomes
as the cell approached mitosis. In addition, it was long established that introduction of extra centrioles into eggs through
polyspermic fertilization led to supernumerary centrosomes
and multipolar divisions.
However, any simple claim that centrioles were the spindle poles or the centrosomal MTOC in animal cells was open
to criticism. Although spindle microtubules converge on the
centriolar region, they do not contact the centrioles themselves but rather end in the pericentriolar material. In vitro
work with disrupted, isolated centrosomes incubated in
tubulin revealed that microtubules grew from bits of pericentriolar material, not naked centrioles [39]. Spindle poles in
animal cells are organized by the centrosomal MTOC (term
coined by [31]). Nevertheless, some felt that centrioles were
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4. Centrosomes versus mitotic poles
why he was so deliberate in doing this, his later writings
suggested that he was thinking about integrating his findings
with spindle assembly in higher plant and acentriolar animal
cells (see [30]). Implicit in his writings and those of others
[31,32] was the notion that all cells, even those of higher
plants, must have something ( presumably an MTOC) at the
spindle poles to originate and/or anchor spindle and kinetochore fibres. In animal cells, this is a corpuscular centrosome,
whereas in ‘acentrosomal’ cells, this something would be
diffuse enough not to be readily apparent. Mazia did not
believe that chromosomes could organize spindles, because
sea urchin zygotes with only one centrosome assembled a
monopolar spindle [29,33]. In addition, zygotes in which pronuclear fusion had been prevented assemble a bipolar spindle
at the sperm pronucleus, but no spindle in association with
the condensed female chromosomes [34,35]. Other supporting
observations (reviewed in [29]) included the classic observation of polar caps in prophase plant cells. These have the
appearance of two cones at the nucleus whose tapered ends
predict the orientation of the anastral spindle after nuclear
envelope breakdown. Mazia thought these polar caps pointed
to the MTOCs that would organize the spindle. In addition, in
some plant and animal cells the spindle starts as a multipolar
figure (polyarchal spindle assembly) that soon resolves into
a bipolar configuration. This bundling of multiple spindle
poles was interpreted to be due to the presence of diffuse
centrosomes not visible by conventional methods.
Mazia pointed out in 1961 that the activity of mitotic
centres was the same whether centrioles were present or not.
Later, he was more explicit: ‘Mitotic centers are real; they are
not abstractions improvised to explain mitosis in the absence
of centrioles.. . .As to centrioles, the problem will be to understand how centers make centrioles, rather than the other way
around.’ [36]. He went on to say ‘In my opinion the phenotypic centrioles are giving accurate information about the
progress of the reproduction of the centrosome that they inhabit’ [29]. These thoughts mirrored those of Pickett-Heaps
[32] who said ‘The centriole is simply one form of the
expression of the informational content of MTOCs in the cell’.
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microsurgery) revealed that second division blastomeres with
monopolar spindles showed prolonged prometaphase and the
monopolar spindles could split to form bipolar spindles that
supported bipolar division. At the third mitosis, the daughter
cells inheriting split poles again assembled monopolar spindles
that in turn could split to form a bipolar spindle during
prolonged prometaphase (figure 1). The same was observed
for the fourth mitosis. These observations revealed that splitting
of poles during prolonged prometaphase was not due to
some peculiar action of mercaptoethanol during first mitosis;
rather it was something that spontaneously occurs when
prometaphase is prolonged—even well after experimental intervention. Spindle pole splitting during prolonged prometaphase
is not peculiar to echinoderm zygotes. When prometaphase is
prolonged in Chinese hamster ovary (CHO) cells with colcemid,
one or both centrosomes can split leading to multipolar
divisions in slightly less than half the cells [22,23].
Although Mazia [18,24] indicated that centrioles could be
morphological correlates for the mitotic centres, this had not
been tested. This was substantiated in a serial section electron
microscopy characterization of individual mercaptoethanoltreated sea urchin zygotes that had been previously followed
in vivo [25]. After release from mercaptoethanol, each pole of
the tetrapolar spindles contained just a single centriole (also
see [23]). At second mitosis, the monopolar spindles were
found to contain a pair of centrioles. After these monopolar
spindles split into bipolar spindles, the poles contained a
single centriole apiece. Thus, the functional behaviour of
mitotic centres is mirrored by the splitting and later duplication of centriole pairs (diagrammed in figure 1). These
results raised the question of whether centrioles were actually
the mitotic centres, a possibility supported by observations
that microinjection of partially isolated sperm centrioles or
Chlamydomonas basal bodies into echinoderm zygotes or frog
eggs led to the assembly of supernumerary asters [26–28].
In addition, nature’s own microinjection ‘experiment’—
namely polyspermy—was long known to lead to the assembly of extra centrosomes. However, this notion that centrioles
were the mitotic centres ran afoul of the need to explain how
higher plant and acentrosomal animal cells assembled bipolar spindles without centrioles. At the time, Mazia felt that
centrioles, when present, were good ‘advertisements’ for
the mitotic centres, not the mitotic centres themselves. Put
differently, he thought that even though the mitotic centres
may be spatially and mechanically associated with centrioles
in most animal cells, they are distinct entities.
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6. Centriole duplication: only one daughter
The numerical and spatial specificity of procentriole assembly
has historically led to the notion that the mother centriole
has only one unique site or ‘template’ that can seed the
assembly of the new procentriole (see [55] for a thoughtful
discussion of the possible meanings for this ambiguous
term). In its simplest form, the term template implies a structure that directly patterns the cartwheel structure and the
nine triplet microtubules of the procentriole—a ‘rubber
stamp’ in the parlance of Fulton. In Paramecium, there is a
plaque next to the parent basal body upon which the barrel
of triplet microtubules progressively assembles [56] and in
Chlamydomonas, there is a looped fibre at the mother basal
body containing nine densely staining foci that later elaborate
into triplet microtubules [57] (reviewed in [58]). However, it
is uncertain whether these structures are the proposed template on the mother basal body or the early assembly
intermediates of daughter basal bodies.
Another possibility is that the template is not a patterning
structure but rather a tiny seed or singularity at the proximal
wall of the mother centrioles that specifies the site where a
daughter centriole self-assembles. In the words of Mazia
[18] ‘. . .a body as complex as the centriole contains a reproducing “germ” or “seed” of molecular dimensions. This gives
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what had long been assumed. Animal cells organize spindle
poles differently. Moreover, the centrosomal and the acentrosomal spindle assembly pathways are not mutually exclusive.
We now know that these pathways normally act in concert
to enhance the timeliness and fidelity of spindle assembly
in animal cells. Indeed, when centrioles are not present in
animal somatic cells, the fidelity of bipolar spindle assembly
and fidelity of chromosome distribution in anaphase are
significantly compromised [49,50].
Thus, for animal cells with canonical centrosomes, we can
now think about how centriole duplication and separation
control centrosome duplication. The notion that centrioles
in animal cells collect and organize the centrosomal MTOC
by being intimately associated with elements of the PCM is
well supported (see [51–54]). Beyond supernumerary aster
formation with polyspermy or injected bare centrioles, functional evidence for this belief came from the finding that
loading HeLa cells with a monoclonal antibody to polyglutamylated tubulin lead to the total disappearance of the
centrioles and the scattering of the PCM [51]. More
recently, subdiffraction-resolution fluorescence microscopy
has revealed cylindrical or ring shaped localizations of various PCM proteins at the outside of the centriole wall—
some consistently closer and others further from the triplet
microtubules [53]. Pericentrin molecules extend in a radial
fashion with their C-terminal PACT domains at the centriole
wall. The specificity of pericentrin attachment to the centriole
wall is suggested by the report that pericentrin has a symmetrical organization reflecting the ninefold symmetry of
the centriole [52] (reviewed in [54]).
The problem of what limits centrosome duplication in
animal cells is now moved down a level to what mechanisms
ensure that only one procentriole is assembled at, but
slightly separated from, the proximal end of each mother centriole. This has been one of the enduring mysteries in the
centrosome field.
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not necessarily irrelevant; Don Fawcett [40] said ‘This does
not necessarily mean that the centrioles are inactive during
cell division. They may control aggregation of the pericentriolar material or may be involved in its activation at the
appropriate time in the cell cycle’.
Other investigators, however, had a very different and
provocative point of view. They proposed that centrioles
were passive or inert passengers attached to the spindle
poles. In their view, the spindle leads the centrioles to its
poles and their presence there is a consequence, not a cause,
of spindle assembly. This is simply the animal cell’s way of
ensuring the equal partitioning of basal bodies to daughter
cells—much like the chromosomes [32,41]. In spindle pole formation, the MTOC is the key player, be it focal in animal cells
or diffuse—even invisible—in higher plant cells. Significantly,
studies by Roland Dietz reported for primary crane fly spermatocytes that the centrioles could be displaced from the
late diakinesis nucleus by flattening the cell; nevertheless,
such cells assembled functional bipolar spindles (reviewed
in [42]). Furthermore, exacting electron microscopy of such
cells did not find accumulations of pericentriolar material
(PCM) at these spindle poles; the PCM stayed with the centrioles [42]. In addition, for spermatocyte meiosis in the
silkworm moth Bombyx mori the centrioles were not associated
with astral fibres and were substantially separated from the
spindle poles at metaphase [41]. So, even animal cells that normally have centrioles do not need them for bipolar spindle
assembly, a conclusion borne out by later work [43–45].
These arguments, however, begged the question of what
precisely controlled the number of spindle poles in acentrosomal cells. In grappling with this issue, Mazia [29,33] turned
back to MTOCs as the determinant of spindle poles (also
see [32]). He proposed in 1987 that ‘The concept of the centrosome can be generalized and unified by abandoning the old
definitions that called for a corpuscular body with a constant
morphology. We can, instead, regard the centrosome as a
flexible, ultimately linear body which can take on different
shapes in different cells. . .’. He modelled the centrosome as
a linear fibre with microtubule nucleating sites along its
length. He thought that plant cells at mitosis have flat and
diffuse centrosomes while animal cells have condensed
fibres (much like mitotic chromosomes). Perhaps each centriole could simply be 27 microtubule nucleating sites along
the fibre wrapped up in a circular pattern! (D. Mazia 1987,
personal communication). In 1984, he said ‘Once we establish
the flexibility of centrosomes,. . .we can understand why
some centrosomes, in extended forms, might be hard to find’.
As to what determined centrosome copy number, he implied
that this was determined by the number of linear threads;
each spindle pole would contain a pair of threads—matching
the number of mitotic centres [33].
At about this time, change was on the horizon. An important, but not fully recognized, contribution to the thinking of
the centrosome field came from a body of work demonstrating bipolar spindle assembly based on microtubule assembly
around chromatin, along with microtubule sorting/bundling
by plus and minus end-directed microtubule motor proteins
[46–48]. The demonstration of strictly chromatin-based
bipolar spindle assembly around chromosomes freed the centrosome field from the baggage of having to coordinately
explain spindle pole formation in animal and plant cells. Acentrosomal cells can build a bipolar spindle without having
something, such as an MTOC, at spindle poles, contrary to
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However, a number of observations challenge the
template or singularity hypothesis. De novo centriole assembly has been demonstrated for a wide variety of cell types,
including parthenogenetically activated sea urchin eggs
[75], mammalian somatic cells [45,76], Chlamydomonas [77],
and certain insect eggs when sperm centrioles are not present
[70,78] (reviewed in [79]). The presence of a mother centriole
and its putative singularity are clearly not needed for the
assembly of new centrioles. Another challenge is the experimentally induced assembly of several procentrioles around
each mother centriole in cultured cells which argues against
the notion that a mother centriole can bear only one singularity or template in any given cell cycle [72,73,80]. A direct
challenge comes from the finding that laser ablation of a
daughter centriole in S-arrested HeLa and CHO cells results
in the formation of a new procentriole at the mother centriole,
not always at the exact location of the previous procentriole
[81]. Some ablations even took out a bit of the mother centriole which should have removed the putative template.
Although S phase is constitutively permissive for procentriole
assembly, the presence of a daughter centriole blocks further
daughter assembly at that mother centriole. New centriole
assembly appears to be controlled at the level of the centrioles
and surrounding PCM. Consistent with this notion, increasing the size of the PCM by pericentrin overexpression
allows assembly of multiple procentrioles near the mother
centriole [81].
If there is no template on the mother centriole, we are left
with a messier notion that the PCM together with proteins on
the wall of the mother centriole provides a small local environment that promotes the self-assembly of the daughter
centriole [81] (also see [70]). This begs the question of how
such a permissive microenvironment can be precisely controlled to yield the formation of only one procentriole at
each mother with high fidelity in the face of non-limiting
pools of centriolar subunits. Perhaps the subunit and regulatory kinase pools in the microenvironment are so limited that
there is only enough ‘stuff’ to make one daughter.
Although the concept of a template or singularity at the
mother centriole appeared to be moribund, recent work has
breathed new life into this idea. Super-resolution immunofluorescence microscopy revealed that Plk4, a kinase
necessary for procentriole initiation, is localized to a single
small spot on the wall of the mother centriole throughout
the cell cycle [61]. This spot colocalizes with Sas-6 as the procentriole starts assembling. This plus the finding that the Plk4
spot is present in G1 before Sas-6 presence indicate that this
spot could be the singularity where only one procentriole
can assemble at each mother centriole.
If so, we are back to almost full circle to thinking about a
pre-existing singularity or initiating complex at each mother
centriole that seeds procentriole assembly. The observations
that previously argued against a singularity may simply tell
us that this one Plk4-containing spot is not determined by
an autonomous SRE; there must be different mechanisms in
play. Rather than seeing a discrepancy, we can use these
observations critical of a singularity as valuable insights
into the functional properties of the system that lead to the
formation of a singularity. For example, we could consider
the possibility that a tightly regulated equilibrium of regulatory kinases, phosphatases and structural proteins in the
microenvironment at the centriole wall supports the formation of centriole initiating complexes with kinetics slow
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rise to its like, which in turn direct the growth of a replica of
the original body’. This is what Fulton [55] calls a ‘selfreplicating entity’ (SRE) that is conceptually distinct from
the morphological barrel of nine triplet microtubules. Needless to say, the duplication of this proposed singularity
would have to be under tight control to ensure that only
one is present at each mother centriole at each cell cycle. Support for this notion has come from images of a focus of
gamma tubulin on the mother centriole from which the
microtubules of the procentriole grow [59,60] (also see
Sonnen [61]). Functional evidence for the existence of such
an SRE came from the finding that the amoeboid form of
Naegleria propagates without centrioles but when stressed,
this organism differentiates into a flagellated motile form
with the assembly of just two basal bodies (reviewed in
[55]). More recently, Collins [62] reported that when assembly
of cytoplasmic microtubules and new centriolar triplet microtubules is completely blocked, there is a time-dependent
accumulation of precursors that soon elaborate into multiple
centrioles after removal of the microtubule inhibitor.
The nature of this proposed self-replicating ‘seed’ was a
mystery. Almost 50 years ago, researchers started considering
the notion that centrioles, such as mitochondria and chloroplasts, have their own DNA that would serve a local
genomic function (see Hall [63] and references therein). Conceivably, the duplication of this DNA would produce a copy
around which only one procentriole assembles at each mother
centriole. A variant on this theme held that centrioles, such as
ribosomes, contain RNA that serves a structural role that
could in principle limit the number of daughter centrioles
assembled at each cell cycle. These possibilities inspired
numerous studies, some of which sought to demonstrate
the localization of DNA (or RNA) to centrioles/centrosomes
on the assumption that presence indicates function. In the
end, these studies have been regarded as inconclusive due
to technical limitations and an inability to rule out more mundane explanations, such as the redistribution and differential
extraction of nuclear and cytoplasmic nucleic acids. More
interesting functional studies reported that the aster inducing
activity of injected centrioles or basal bodies was nuclease
sensitive. These also were subjected to serious technical concerns that have taken their conclusions out of serious
consideration. Another promising lead was the report of
RNAase/pronase sensitive fibrogranular inclusions in the
lumen of ciliate basal bodies [64]. Later investigation revealed
that these luminal structures were alpha-amylase sensitive,
indicating that they were glycogen inclusions [65]. Reviews
of this subject are found in [17,55,66,67].
The ongoing appeal of the template or singularity
hypothesis has come from observations that only one procentriole assembles at each mother despite abundant and
complete cytoplasmic pools of centriolar subunits. For
example, early sea urchin and frog zygotes contain complete
pools of subunits on hand at fertilization to make many centrioles [68,69]. The same holds true for early Drosophila
embryos that are reported to have subunit pools sufficient
to assemble 2 1013 centriole pairs [70]. Even mammalian
somatic cells contain enough centriolar subunits to assemble
multiple procentrioles within a single cell cycle when procentriole initiating proteins, such as Plk4 kinase, STIL or
SAS-6, are overexpressed [71–74]. There are adequate pools
of all other centriolar subunits to match the activity of the
individually overexpressed centriole initiating proteins.
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Acknowledgements. I thank Dr Yumi Uetake for substantial help collecting and formatting the references. I also pay homage to Daniel
Mazia for his influence on the thinking of the centrosome field.
Conversations with him were never linear; many issues would be
discussed—rarely with resolution. Going back to his writings, with
the advantage of hindsight, one can find structure behind his ramblings and a nuanced approach. He had a remarkable ability to
step back from the forest of observations to seek basic principles.
Lastly, I sincerely apologize to those whose work was not described
or referenced due to space limitations.
Funding statement. Studies of the author referenced in this review were
supported by NIH GM30758.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Bornens M. 2012 The centrosome in cells and
organisms. Science 335, 422–426. (doi:10.1126/
science.1209037)
Brinkley BR. 2001 Managing the centrosome
numbers game: from chaos to stability in cancer cell
division. Trends Cell Biol. 11, 18 –21. (doi:10.1016/
S0962-8924(00)01872-9)
Ganem NJ, Godinho SA, Pellman D. 2009
A mechanism linking extra centrosomes to
chromosomal instability. Nature 460, 278 –282.
(doi:10.1038/nature08136)
Crasta K et al. 2012 DNA breaks and chromosome
pulverization from errors in mitosis. Nature 482,
53 –58. (doi:10.1038/nature10802)
Orr-Weaver TL, Weinberg RA. 1998 A checkpoint on
the road to cancer. Nature 392, 223 –224. (doi:10.
1038/32520)
Nigg EA. 2002 Centrosome aberrations: cause or
consequence of cancer progression? Nat. Rev. Cancer
2, 815–825. (doi:10.1038/nrc924)
Lengauer C, Kinzler KW, Vogelstein B. 1998 Genetic
instabilities in human cancers. Nature 396,
643–649. (doi:10.1038/25292)
D’Assoro AB, Lingle WL, Salisbury JL. 2002
Centrosome amplification and the development of
cancer. Oncogene 21, 6146 –6153. (doi:10.1038/sj.
onc.1205772)
Goepfert TM, Adigun YE, Zhong L, Gay J, Medina D,
Brinkley WR. 2002 Centrosome amplification and
overexpression of aurora A are early events in rat
mammary carcinogenesis. Cancer Res. 62, 4115–4122.
Gall JG. 2004 Early studies on centrioles and centrosomes,
pp. 3–15. Weinheim, Germany: Wiley-VCH.
Wilson E. 1925 The cell in development and heredity.
New York, NY: Macmillan.
Van Beneden E, Neyt A. 1887 Nouvelles recherches sur
la maturation de l’oeuf, la fécondation et la division
cellulaire. Bull. Acad. R. Sci. Belg. 147, 215–298.
Wilson E. 1896 The cell in development and
inheritance. New York, NY: Macmillan.
Boveri T. 2008 Concerning the origin of malignant
tumours by Theodor Boveri. Translated and
annotated by Henry Harris. J. Cell Sci. 121(Suppl. 1),
1– 84. (doi:10.1242/jcs.025742)
Maderspacher F. 2008 Theodor Boveri and the
natural experiment. Curr. Biol. 18, R279 –R286.
(doi:10.1016/j.cub.2008.02.061)
16. Wheatley D. 1982 The centriole: a central enigma of
cell biology. Amsterdam, The Netherlands: Elsevier
Biomedical Press.
17. Sapp J. 1998 Freewheeling centrioles. Hist. Philos.
Life Sci. 20, 255–290.
18. Mazia D, Harris PJ, Bibring T. 1960 The multiplicity
of the mitotic centers and the time-course of their
duplication and separation. J. Biophys. Biochem.
Cytol. 7, 1–20. (doi:10.1083/jcb.7.1.1)
19. Mazia D, Zimmerman AM. 1958 SH compounds in
mitosis. II. The effect of mercaptoethanol on the
structure of the mitotic apparatus in sea urchin
eggs. Exp. Cell Res. 15, 138–153. (doi:10.1016/
0014-4827(58)90070-3)
20. Sluder G. 2004 Centrosome duplication and its
regulation in the higher animal cell. In Centrosome in
development and disease (ed. EA Nigg), pp. 167–189.
Weinheim, Germany: Wiley-VCH.
21. Sluder G, Begg DA. 1985 Experimental analysis of
the reproduction of spindle poles. J. Cell Sci. 76,
35 –51.
22. Stubblefield E. 1967 Centriole replication in a
mammalian cell. pp. 175– 193. Baltimore, MD:
Williams and Wilkins.
23. Keryer G, Ris H, Borisy GG. 1984 Centriole
distribution during tripolar mitosis in Chinese
hamster ovary cells. J. Cell Biol. 98, 2222–2229.
(doi:10.1083/jcb.98.6.2222)
24. Mazia D. 1961 Mitosis and the physiology of cell
division. San Diego, CA: Academic Press.
25. Sluder G, Rieder CL. 1985 Centriole number and the
reproductive capacity of spindle poles. J. Cell Biol.
100, 887 –896. (doi:10.1083/jcb.100.3.887)
26. Hamaguchi Y, Kuriyama R. 1982 Aster formation in
sand dollar eggs by microinjection of calcium
buffers and centriolar complexes isolated from
starfish sperm. Exp. Cell Res. 141, 450–454.
(doi:10.1016/0014-4827(82)90233-6)
27. Heidemann SR, Kirschner MW. 1975 Aster formation
in eggs of Xenopus laevis. Induction by isolated
basal bodies. J. Cell Biol. 67, 105 –117. (doi:10.
1083/jcb.67.1.105)
28. Maller J, Poccia D, Nishioka D, Kidd P, Gerhart J,
Hartman H. 1976 Spindle formation and cleavage in
Xenopus eggs injected with centriole-containing
fractions from sperm. Exp. Cell Res. 99, 285–294.
(doi:10.1016/0014-4827(76)90585-1)
29. Mazia D. 1984 Centrosomes and mitotic poles.
Exp. Cell Res. 153, 1 –15. (doi:10.1016/00144827(84)90442-7)
30. Szollosi A, Ris H, Szollosi D, Debec A. 1986 A
centriole-free Drosophila cell line. A high voltage EM
study. Eur. J. Cell Biol. 40, 100– 104.
31. Pickett-Heaps JD. 1969 The evolution of the mitotic
apparatus: an attempt at comparative ultrastructural
cytology in dividing plant cells. Cytobios 3, 257–280.
32. Pickett-Heaps JD. 1971 The autonomy of the
centriole: fact or fallacy? Cytobios 3, 205–214.
33. Mazia D. 1987 The chromosome cycle and the
centrosome cycle in the mitotic cycle. Int. Rev. Cytol.
100, 49– 92.
34. Sluder G, Rieder CL. 1985 Experimental separation
of pronuclei in fertilized sea urchin eggs:
chromosomes do not organize a spindle in the
absence of centrosomes. J. Cell Biol. 100, 897 –903.
(doi:10.1083/jcb.100.3.897)
35. Sluder G, Miller FJ, Rieder CL. 1989 Reproductive
capacity of sea urchin centrosomes without
centrioles. Cell Motil. Cytoskeleton 13, 264–273.
(doi:10.1002/cm.970130405)
36. Mazia D. 1978 Origin of twoness in cell reproduction.
San Diego, CA: Academic Press.
37. Brinkley BR, Stubblefield E, Hsu TC. 1967 The effects
of colcemid inhibition and reversal on the fine
structure of the mitotic apparatus of Chinese
hamster cells in vitro. J. Ultrastruct. Res. 19, 1–18.
(doi:10.1016/S0022-5320(67)80057-1)
38. Stubblefield E, Brinkley BR. 1967 Architecture and
function of the mammalian centriole. Symp. Int.
Soc. Cell Biol. 6, 175 –218.
39. Gould RR, Borisy GG. 1977 The pericentriolar
material in Chinese hamster ovary cells nucleates
microtubule formation. J. Cell Biol. 73, 601–615.
(doi:10.1083/jcb.73.3.601)
40. Fawcett DW. 1981 Centrioles, pp. 551–574,
2nd edn. Philadelphia, PA: W B Saunders.
41. Friedlander M, Wahrman J. 1970 The spindle as a basal
body distributor. A study in the meiosis of the male
silkworm moth, Bombyx mori. J. Cell Sci. 7, 65–89.
42. Steffen W, Fuge H, Dietz R, Bastmeyer M, Muller G.
1986 Aster-free spindle poles in insect
spermatocytes: evidence for chromosome-induced
spindle formation? J. Cell Biol. 102, 1679–1687.
(doi:10.1083/jcb.102.5.1679)
Phil. Trans. R. Soc. B 369: 20130455
References
7
rstb.royalsocietypublishing.org
enough that only one forms at a time. Once formed, the
newly assembled complex could have an activity that
immediately inhibits the formation of other initiating complexes. Overexpression of centriole initiating proteins could
perturb this putative equilibrium, so that multiple procentrioles are assembled at once. After laser ablation of
the daughter centriole, a new Plk4-containing spot could
form as it would under normal circumstances. Obviously,
there is much still to be learned and our fascination with
how centriole duplication is so precisely regulated will
continue unabated.
Downloaded from http://rstb.royalsocietypublishing.org/ on June 14, 2017
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
detectable cell cycle. J. Cell Biol. 110, 2033–2042.
(doi:10.1083/jcb.110.6.2033)
Rodrigues-Martins A, Riparbelli M, Callaini G, Glover
DM, Bettencourt-Dias M. 2007 Revisiting the role of
the mother centriole in centriole biogenesis.
Science 316, 1046 – 1050. (doi:10.1126/science.
1142950)
Habedanck R, Stierhof YD, Wilkinson CJ, Nigg EA.
2005 The Polo kinase Plk4 functions in centriole
duplication. Nat. Cell Biol. 7, 1140–1146. (doi:10.
1038/ncb1320)
Kleylein-Sohn J, Westendorf J, Le Clech M,
Habedanck R, Stierhof YD, Nigg EA. 2007 Plk4induced centriole biogenesis in human cells.
Dev. Cell 13, 190 –202. (doi:10.1016/j.devcel.2007.
07.002)
Strnad P, Leidel S, Vinogradova T, Euteneuer U,
Khodjakov A, Gonczy P. 2007 Regulated HsSAS-6
levels ensure formation of a single procentriole per
centriole during the centrosome duplication cycle.
Dev. Cell 13, 203 –213. (doi:10.1016/j.devcel.2007.
07.004)
Arquint C, Sonnen KF, Stierhof YD, Nigg EA. 2012
Cell-cycle-regulated expression of STIL controls
centriole number in human cells. J. Cell Sci. 125,
1342– 1352. (doi:10.1242/jcs.099887)
Kallenbach RJ, Mazia D. 1982 Origin and maturation
of centrioles in association with the nuclear
envelope in hypertonic-stressed sea urchin eggs.
Eur. J. Cell Biol. 28, 68– 76.
La Terra S, English CN, Hergert P, McEwen BF,
Sluder G, Khodjakov A. 2005 The de novo centriole
assembly pathway in HeLa cells: cell cycle
progression and centriole assembly/maturation.
J. Cell Biol. 168, 713–722. (doi:10.1083/jcb.
200411126)
Marshall WF. 2001 Centrioles take center stage.
Curr. Biol. 11, R487 –R496. (doi:10.1016/S09609822(01)00289-5)
Peel N, Stevens NR, Basto R, Raff JW. 2007
Overexpressing centriole-replication proteins in vivo
induces centriole overduplication and de novo
formation. Curr. Biol. 17, 834–843. (doi:10.1016/j.
cub.2007.04.036)
Debec A, Sullivan W, Bettencourt-Dias M. 2010
Centrioles: active players or passengers during
mitosis? Cell Mol. Life Sci. 67, 2173– 2194. (doi:10.
1007/s00018-010-0323-9)
Duensing A, Spardy N, Chatterjee P, Zheng L, Parry
J, Cuevas R, Korzeniewski N, Duensing S. 2009
Centrosome overduplication, chromosomal
instability, and human papillomavirus oncoproteins.
Environ. Mol. Mutagen 50, 741–747. (doi:10.1002/
em.20478)
Loncarek J, Hergert P, Magidson V, Khodjakov A.
2008 Control of daughter centriole formation by the
pericentriolar material. Nat. Cell Biol. 10, 322 –328.
(doi:10.1038/ncb1694)
8
Phil. Trans. R. Soc. B 369: 20130455
55. Fulton C. 1971 Centrioles, pp. 170– 213. New York,
NY: Springer.
56. Dippell RV. 1968 The development of basal bodies
in paramecium. Proc. Natl Acad. Sci. USA 61,
461 –468. (doi:10.1073/pnas.61.2.461)
57. Gould RR. 1975 The basal bodies of Chlamydomonas
reinhardtii. Formation from probasal bodies,
isolation, and partial characterization. J. Cell Biol.
65, 65 –74. (doi:10.1083/jcb.65.1.65)
58. Beisson J, Wright M. 2003 Basal body/centriole
assembly and continuity. Curr. Opin. Cell Biol.
15, 96 –104. (doi:10.1016/S0955-0674(02)00017-0)
59. Guichard P, Chretien D, Marco S, Tassin AM. 2010
Procentriole assembly revealed by cryo-electron
tomography. EMBO J. 29, 1565 –1572. (doi:10.
1038/emboj.2010.45)
60. Fuller SD, Gowen BE, Reinsch S, Sawyer A, Buendia
B, Wepf R, Karsenti E. 1995 The core of the
mammalian centriole contains gamma-tubulin.
Curr. Biol. 5, 1384 –1393. (doi:10.1016/S09609822(95)00276-4)
61. Sonnen KF, Schermelleh L, Leonhardt H, Nigg EA.
2012 3D-structured illumination microscopy provides
novel insight into architecture of human
centrosomes. Biol. Open 1, 965– 976. (doi:10.1242/
bio.20122337)
62. Collins ES, Hornick JE, Durcan TM, Collins NS, Archer
W, Karanjeet KB, Vaughan KT, Hinchcliffe EH. 2010
Centrosome biogenesis continues in the absence of
microtubules during prolonged S-phase arrest.
J. Cell Physiol. 225, 454 –465. (doi:10.1002/
jcp.22222)
63. Hall JL, Ramanis Z, Luck DJ. 1989 Basal body/
centriolar DNA: molecular genetic studies in
Chlamydomonas. Cell 59, 121–132. (doi:10.1016/
0092-8674(89)90875-1)
64. Dippell RV. 1976 Effects of nuclease and protease
digestion on the ultrastructure of paramecium basal
bodies. J. Cell Biol. 69, 622–637. (doi:10.1083/jcb.
69.3.622)
65. Mignot JP, Brugerolle G, Didier P, Bornens M. 1993
Basal-body-associated macromolecules: a continuing
debate. Trends Cell Biol. 3, 220–223. (doi:10.1016/
0962-8924(93)90115-H)
66. Johnson KA, Rosenbaum JL. 1991 Basal bodies and
DNA. Trends Cell Biol. 1, 145 –149. (doi:10.1016/
0962-8924(91)90002-Q)
67. Marshall WF, Rosenbaum JL. 2000 Are there nucleic
acids in the centrosome? Curr. Top. Dev. Biol. 49,
187 –205.
68. Sluder G, Miller FJ, Cole R, Rieder CL. 1990 Protein
synthesis and the cell cycle: centrosome
reproduction in sea urchin eggs is not under
translational control. J. Cell Biol. 110, 2025–2032.
(doi:10.1083/jcb.110.6.2025)
69. Gard DL, Hafezi S, Zhang T, Doxsey SJ. 1990
Centrosome duplication continues in cycloheximidetreated Xenopus blastulae in the absence of a
rstb.royalsocietypublishing.org
43. Khodjakov A, Cole RW, Oakley BR, Rieder CL. 2000
Centrosome-independent mitotic spindle formation
in vertebrates. Curr. Biol. 10, 59 –67. (doi:10.1016/
S0960-9822(99)00276-6)
44. Basto R, Lau J, Vinogradova T, Gardiol A, Woods CG,
Khodjakov A, Raff JW. 2006 Flies without centrioles.
Cell 125, 1375–1386. (doi:10.1016/j.cell.2006.05.025)
45. Uetake Y, Loncarek J, Nordberg JJ, English CN, La
Terra S, Khodjakov A, Sluder G. 2007 Cell cycle
progression and de novo centriole assembly after
centrosomal removal in untransformed human cells.
J. Cell Biol. 176, 173– 182. (doi:10.1083/jcb.
200607073)
46. Karsenti E, Newport J, Hubble R, Kirschner M. 1984
Interconversion of metaphase and interphase
microtubule arrays, as studied by the injection of
centrosomes and nuclei into Xenopus eggs. J. Cell
Biol. 98, 1730 –1745. (doi:10.1083/jcb.98.5.1730)
47. Heald R, Tournebize R, Blank T, Sandaltzopoulos R,
Becker P, Hyman A, Karsenti E. 1996 Selforganization of microtubules into bipolar spindles
around artificial chromosomes in Xenopus egg
extracts. Nature 382, 420–425. (doi:10.1038/
382420a0)
48. Walczak CE, Vernos I, Mitchison TJ, Karsenti E, Heald
R. 1998 A model for the proposed roles of different
microtubule-based motor proteins in establishing
spindle bipolarity. Curr. Biol. 8, 903–913. (doi:10.
1016/S0960-9822(07)00370-3)
49. Hornick JE, Mader CC, Tribble EK, Bagne CC,
Vaughan KT, Shaw SL, Hinchcliffe EH. 2011
Amphiastral mitotic spindle assembly in vertebrate
cells lacking centrosomes. Curr. Biol. 21, 598–605.
(doi:10.1016/j.cub.2011.02.049)
50. Sir JH, Putz M, Daly O, Morrison CG, Dunning M,
Kilmartin JV, Gergely F. 2013 Loss of centrioles
causes chromosomal instability in vertebrate
somatic cells. J. Cell Biol. 203, 747–756. (doi:10.
1083/jcb.201309038)
51. Bobinnec Y, Khodjakov A, Mir LM, Rieder CL, Edde
B, Bornens M. 1998 Centriole disassembly in vivo
and its effect on centrosome structure and function
in vertebrate cells. J. Cell Biol. 143, 1575 –1589.
(doi:10.1083/jcb.143.6.1575)
52. Mennella V, Keszthelyi B, McDonald KL, Chhun B,
Kan F, Rogers GC, Huang B, Agard DA. 2012
Subdiffraction-resolution fluorescence microscopy
reveals a domain of the centrosome critical for
pericentriolar material organization. Nat. Cell Biol.
14, 1159 –1168. (doi:10.1038/ncb2597)
53. Fu J, Glover DM. 2012 Structured illumination of the
interface between centriole and peri-centriolar
material. Open Biol. 2, 120104. (doi:10.1098/rsob.
120104)
54. Mennella V, Agard DA, Huang B, Pelletier L. 2013
Amorphous no more: subdiffraction view of the
pericentriolar material architecture. Trends Cell Biol.
24, 188–197. (doi:10.1016/j.tcb.2013.10.001)