2697
Journal of Cell Science 111, 2697-2706 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS5008
COMMENTARY
Centrosomes and microtubule organisation during Drosophila development
Cayetano González*, Gaia Tavosanis and Cristiana Mollinari
European Molecular Biology Laboratory Meyerhofstrasse 1, 69117 Heidelberg, Germany
*Author for correspondence (e-mail: [email protected])
Published on WWW 27 August 1998
SUMMARY
Are the microtubule-organising centers of the different cell
types of a metazoan interchangeable? If not, what are the
differences between them? Do they play any role in the
differentiation processes to which these cells are subjected?
Nearly one hundred years of centrosome research has
established the essential role of this organelle as the main
microtubule-organising center of animal cells. But only
now are we starting to unveil the answers to the challenging
questions which are raised when the centrosome is studied
within the context of a pluricellular organism. In this
review we present some of the many examples which
illustrate how centrosomes and microtubule organisation
changes through development in Drosophila and discuss
some of its implications.
INTRODUCTION
differentiated cells account for the vast majority of the cells
which are present in post-embryonic stages.
We have selected for discussion only some of the many
examples which illustrate the specific subject of microtubule
organisation changes through development in Drosophila. For
reviews on the biochemistry and genetics of centrosomes in
Drosophila and other organisms the reader is referred to Fulton
(1971); Mazia (1984); Kimble and Kuriyama (1992); Kalnins
(1992); Kalt and Schliwa (1993); Kellog et al. (1994);
Gonzalez et al. (1994); Balczon (1996); and Paoletti and
Bornens (1997). For thorough discussions on centrosome
definition and terminology see Fulton (1971); Bornens (1992)
and Paoletti and Bornens (1997). For a comparative study of
centrosomal structures in various species see Mignot (1996).
We use the term centrosome to refer to the cellular organelle
made of two centrioles and pericentriolar material (PCM). In
those cases in which microtubule organisation is driven by
structures different from a classical centrosomes we refer to
them by the more generic term, microtubule organising centers
(MTOCs). Most of what we have learned about MTOCs we
owe to the power of well established experimental model
systems like tissue culture cells, algae, frog egg extracts, and
genetically amenable yeast and fungi (Balczon, 1996).
Therefore, our current view is a limited one in as much as it is
restricted to the centrosomes of single proliferative cell types.
In all these systems, a centrosome organises the cytoskeleton
during interphase, replicates at some stage to produce two
centrosomes, these segregate from one another to define the
poles of the mitotic spindle and, after mitosis, each of them
will organise the interphase cytoskeleton, just as their mother
centrosome did. It is clear, therefore, that to broaden our view
we must expand our interest to include a larger variety of
proliferative and non-proliferative tissues. Metazoans display
many kinds of cell division which have their own cytological
landmarks. Meiosis has many features which make it different
from mitosis, and the two meiotic cell divisions are
significantly different from one another. Moreover, in most
organisms, male and female meiosis are dramatically different.
Even within the purely mitotic kind of cell division, variation
is the rule. In the canonical mitosis, one cell generates two
identical sisters. However, many stem cells divide
asymmetrically to generate two morphologically and
developmentally different daughters. And microtubule
organisation is not limited to proliferating tissues. Non-mitotic
Key words: Centriole, Centrosome, Drosophila, MTOC,
Cytoskeleton, Spindle
CENTROSOME CHANGES AT THE MOLECULAR
LEVEL
Although the number of integral centrosomal components
which have been characterised in detail is still very limited it is
already clear that both the function and expression of some of
them are under developmental control. One of these is CP190,
previously known as Bx63 or DMAP190 (Whitfield et al., 1988;
Kellogg et al., 1989). In mitotic cells, CP190 displays a cellcycle dependent localisation: nuclear during interphase and
centrosomal during mitosis (Frasch et al., 1986; Withfield et al.,
1988). Its behaviour is very different during meiosis. In meiotic
spermatocytes CP190 is dispersed around each spindle in small
dots which are not focused at the spindle poles (Casal et al.,
1990). None of the post-meiotic MTOCs which occur during
spermatogenesis, including the sperm basal body, contain
2698 C. González, G. Tavosanis and C. Mollinari
CENTRIOLE ULTRASTRUCTURE
The oldest and in some cases the most compelling evidence of
developmentally regulated centrosomal plasticity is provided
by ultrastructural studies. In a typical animal cell in G1 the
centrosome consists of a pair of centrioles and a cloud of
pericentriolar material from which microtubules nucleate
(Vandre and Borisy, 1989). The centrioles are highly organised
organelles whose unique features make them readily
identifiable by ultrastructural analysis. In most vertebrate cell
lines, the basic centriolar structure is a cylinder, about 0.2 µm
wide and 0.4 µm long, whose wall is made of nine triplets of
microtubules. The two centrioles of a pair and the proximal and
distal ends of a given centriole are clearly distinguishable by
the ultrastructure of their triplets, the connection between them,
and their association with external appendages (reviewed by
Lange and Gull, 1996). Centriole ultrastructure in Drosophila
is rather variable throughout development. Some of these
changes are summarised in Fig. 1.
Kc23 Cells
Distal
D
P
D
P
D
Applar Spermatocyte
Drosophila
Embryos
Proximal
P
D
Vertebrates
detectable amounts of CP190. During female meiosis CP190
has only been observed in the aster between the second meiotic
spindles (Riparbelli and Callaini, 1996). At the same time, two
spots of CP190 become distinguishable in the sperm aster as
well. The significance of such localisations must be interpreted
with caution since they are sensitive to treatment with
colchicine (Riparbelli et al., 1997). CP60, a mitotic centrosomal
protein whose pattern of expression and subcellular localisation
are very similar to those of CP190 (Kellog et al., 1989), is also
absent in the female meiotic spindle (Matthies et al., 1996).
Another developmentally regulated centrosomal protein of
Drosophila is γ-tubulin. Gamma-tubulin is a highly conserved
member of the tubulin superfamily that plays a major role in
microtubule polymerisation (reviewed by Oakley, 1992). There
are two γ-tubulin genes in Drosophila. The γTUB23C isoform
is essentially ubiquitous. Mutation in this gene impairs viability
and disrupts microtubule organisation during mitosis and male
meiosis (Sunkel et al., 1995; P. Sampaio, C. E. Sunkel and C.
Gonzalez, unpublished). In contrast, the expression of
γTUB37C is restricted to ovaries and early embryos and the
only phenotypic trait associated with mutation in this gene is
female sterility (Tavosanis et al., 1997). The meiotic spindle of
mutant females and the mitotic spindles of their eggs are
severely disrupted (Tavosanis et al., 1997). By
immunofluorescence γ-tubulin has been observed in the middle
of the first meiotic spindle during anaphase and in the aster
which connects the two second meiotic spindles, but not at the
spindle poles (Riparbelli and Callaini, 1996, 1998). Oogenesis,
and the microtubule rearrangements associated with it proceed
normally in individuals mutant for either of these isoforms, but
is disrupted in double mutant females (G. Tavosanis and C.
Gonzalez, unpublished). Thus, γTUB23C is required in somatic
and male germ-line cells, γTUB37C is essential to organise the
female meiotic and the early embryo mitotic spindles and either
of these isoforms is sufficient to provide γ-tubulin function
during oogenesis. There are also two isoforms of cnn, another
integral component of mitotic centrosomes in Drosophila
(Heuer et al., 1995; Li and Kaufman, 1996). Like γ-tubulin, cnn
is associated with every major MTOC during multiple stages of
development including the centrosomes of somatic cells,
spermatocytes and basal bodies. One of the two isoforms of this
gene is produced in testis via alternative splicing (Li et al.,
1998). The significance of this testis isoform, however, is
unclear because the embryonic isoform can restore fertility to
male sterile cnn mutants, suggesting functional redundancy.
There are few other centrosomal proteins of Drosophila that
have been characterised at the molecular level, like LK6 (Kidd
and Raff, 1997) and PP4 (Helps et al., 1998), but they have not
yet been studied in terms of their association with different
MTOCs. Likewise, there are many mutants which disrupt
microtubule organisation, but their molecular cloning is still in
progress. The phenotypes of these mutants range from the
general alteration of microtubule organisation in many tissues
to the very specific disruption of a particular kind of MTOC.
The molecular characterisation of these genes will be essential
to sustantiate MTOC plasticity and its developmental relevance.
P
Fig. 1. Schematic view of centrioles from three different cell types of
Drosophila. The figures on the left represent longitudinal sections.
The figures on the right correspond to transversal sections through
the proximal (P) and distal (D) ends observed from the proximal and
distal side, respectively. The actual number of microtubules present
in the peripheral structures of the embryonic centrioles is still
unclear. While Callaini and Riparbelli (1990) reported triplets,
Callaini et al. (1997) described them as doublets and Moritz et al.
(1995) have reported them to be single microtubules. The
corresponding structure of a typical mature centriole from a
vertebrate cell is also included for comparison. Only microtubules
and the links between them have been depicted. Intraluminal
material, satellites and appendages have been omitted. See text for a
more detailed description and references.
MTOCs in development 2699
THE SHORT CENTRIOLES OF THE EARLY EMBRYO
During embryogenesis, centrioles are considerably shorter than
the mammalian organelles, between 0.18 and 0.2 µm.
Moreover, there are no differences between the proximal and
distal ends of a centriole and between the two centrioles of a
pair. Embryonic mother and daughter centrioles consist of a
typical cartwheel formation with a central microtubule linked
to the nine peripheral microtubule structures by radial spokes.
This cartwheel structure is found along the entire length of the
centriole (Callaini et al., 1997). The actual number of
microtubules present in each of these peripheral structures is
still unclear. In their original study, Callaini and Riparbelli
(1990) reported triplets. A more recent and detailed study
describes them as doublets (Callaini et al., 1997). Moritz et al.
(1995) have reported them to be single microtubules.
The reduced number of peripheral tubules, the cartwheel and
the short length of the cylinder might indicate that the
Drosophila embryonic centriole maintains an immature state.
A simple explanation for this (Moritz et al., 1995) would be
the short length of the early embryonic cell cycles, which occur
without intervening G1 or G2 phases (Foe et al., 1993).
However, the available evidence regarding this point is rather
contradictory. On the one hand, Callaini et al. (1997) have
shown that the apparently ‘immature’ structure is maintained
at later stages of development, when the cell cycle is
considerably longer, like cycles 14 to 16 which display a
distinct G2 and cycle 17 which has both G1 and G2 (Foe et al.,
1993). On the other hand, the ultrastructure of the centriole in
some Drosophila tissue culture cells seems to be significantly
different to that observed in embryonic centrioles. Such is the
case in Kc23 cells, a line derived from embryos, but with a cell
cycle significantly longer than the normal embryonic cell
cycles (Echalier and Ohanessian, 1970). Although still short
and with a cartwheel which spans their entire length, the
centrioles of Kc23 cells have well defined proximal and distal
ends. The distal part is characterised by doublets, while the
proximal end contains triplets, sometimes incomplete for the
third one (Debec and Marcaillou, 1997 and Fig. 1). Thus, in
this case in which cells have a much longer cell cycle the
centrioles achieve a more complex architecture in terms of
proximal-distal differentiation (Fig. 1).
SPERMATOGENESIS: THE EVER CHANGING
CENTRIOLE
During spermatogenesis the centriole changes dramatically in
size and morphology (Tates, 1971; Fuller, 1993). Centriole
duplication in primary spermatocytes results in four identical
centrioles which are 0.9 µm in length, already much longer
than those found in any other Drosophila tissue. These
centrioles display a well defined proximo-distal axis. The
proximal part is made up of nine triplets and a central tubule.
The distal part has no central tubule and the nine peripheral
structures contain one closed and one open tubule (Fig. 1).
During meiosis there appears to be no central tubule in the
centriole. The four centrioles grow until the end of meiosis,
when they reach about 2.6 µm. Most of the growth which
occurs in the early stages takes place in the proximal part while
growth in the older spermatocyte occurs mostly in the distal
end. After meiosis each spermatid has only one centriole which
bears an outgrowth of about 12 µm in the distal part which
corresponds to the future axial filament. The centriole at this
stage has two regions: the caudal segment, which is a
continuation of the axial filament, and the apical segment
which lies in an indentation of the nuclear membrane. By the
end of spermatogenesis the apical segment is 0.8 µm long and
is displaced so that it no longer is positioned inside the nuclear
indentation.
In conclusion, as far as ultra-structure goes, it is very clear
that centrioles are fairly dynamic structures which display
distinct architectural features in different cell lineages and
developmental stages. Since centriole function itself remains
elusive, we do not know what the significance of these
ultrastructural changes may be, but it is tantalising to assume
that such ultrastructural differences may confer specific
functions to MTOCs.
VARIATIONS ON THE CENTROSOME CYCLE
Like centriole ultrastructure, the behaviour of the centrosome
as a whole changes very significantly as the organism develops.
In most animal cell lines, the two centrioles of the centrosome
loose their orthogonal arrangement by the end of G1 and
nucleate procentrioles during S phase (Vandre and Borisy,
1990). Centrosome splitting follows, producing two
centrosomes which contain a centriole pair each. Centrosome
segregation positions the two centrosomes at opposite sites
within the cell, thus defining the location of the mitotic spindle.
After mitosis each daughter cell will inherit one centrosome
with a centriole pair, like the G1 centrosome of the mother cell.
There are notable departures from this theme during
Drosophila development (Fig. 2).
EARLY CENTROSOME REPLICATION IN THE
EMBRYO
During the rapid nuclear cycles which occur in syncytial
blastoderm embryos the centrioles start to lose their orthogonal
arrangement in metaphase, move apart during anaphase and
become widely separated at telophase (Huettner, 1933; Callaini
and Riparbelli, 1990). At the same time, the centrosomal
material expands and flattens, splitting into two units at late
telophase (Warn et al., 1987; Callaini and Riparbelli, 1990).
Thus, each nucleus enters interphase with two separated
centrosomes containing a single centriole from which a
daughter centriole immediately starts to bud. This early
replication and segregation of the centrosomes has often been
interpreted as a consequence of the rapid nuclear cycles and
the absence of intervening gap phases (Foe et al., 1993).
Nevertheless, the reason may not be that simple as more recent
data has shown that the same behaviour can be observed in
cycles 14 to 16. Cycle 17 cells also inherit two separated
centrosomes containing one centriole each, but these do not
bud procentrioles immediately. Whether centriole replication
in these cells occurs later in G1 or in S phase, like it does in
primary spermatocytes for instance, has not yet been
determined.
A distinct property of centrosomes during the syncytial
2700 C. González, G. Tavosanis and C. Mollinari
blastoderm stage is their ability to detach from their nuclei
when nuclear progression is impaired. This was first illustrated
by Freeman et al. (1986) who showed that in embryos derived
from mothers homozygous for gnu, free centrosomes which are
not associated with nuclei can follow several rounds of
replication and eventually, migrate to the cortex. Free
centrosomes have also been observed following the injection
of aphidicolin, an inhibitor of DNA polymerase, into syncytial
embryos (Raff and Glover, 1988; Debec et al., 1996). When
DNA synthesis is inhibited a large number of nuclei fall into
the interior of the embryo while their centrosomes remain in
the cortex. Similar effects have been described for a variety of
mutant conditions (Gonzalez et al., 1990; Sullivan et al., 1990).
ANCHORED CENTROSOMES ORIENT MITOSIS
Oogenesis starts with the asymmetric division of the female
germ-line stem cell to produce a new stem cell and a cystoblast.
The cystoblast then undergoes four rounds of incomplete
mitotic divisions to produce a cyst of 16 cells, the cystocytes,
interconnected by ring canals. One of these cells will become
the oocyte, while the remaining 15 will become nurse cells
(reviewed by Spradling, 1993). Microtubule organisation plays
a crucial role in many steps of this complex process including
the control of the orientation of the mitotic divisions and the
differentiation of the oocyte. The orientation of the mitotic
divisions is mediated by the anchorage of one of the spindle
poles to a membranous organelle known as the
spectrosome/fusome. The spectrosome is found in the stem cell
and the cystoblast and is probably the precursor of the fusome
which is present in the resulting cystocytes. Spectrosome and
fusome contain a network of membranous tubules. Their origin
is unknown, but recent data suggest that they may be a germcell specific modification of smooth endoplasmis reticulum,
Golgi or endosomes (reviewed by McKearin, 1997). The
mitotic spindle of the stem cell is always oriented in such a
way that the resulting stem cell remains located at the tip of
the ovary, while the cystoblast is released posteriorly. This
orientation is mediated by the association of one of the spindle
poles to the spectrosome which is located apically (Lin et al.,
1994; Deng and Lin, 1997). In mutants in which there is no
spectrosome the orientation of the stem cell divisions is
randomised (Deng and Lin, 1997). The orientation of the
following mitosis is mediated by the fusome. Like the
spectrosome during stem cell division, the fusome orientates
oogonial mitosis by anchoring one pole of each mitotic spindle.
This association is thought to be mediated by dynein (McGrail
and Hays, 1997). The correct orientation of these mitotic
divisions appears to be essential for oocyte differentiation
(McGrail and Hays, 1997; de Cuevas et al., 1997). As cell
divisions proceed, the fusome grows and branches through the
ring canals. Shortly after the cyst is formed the fusome retracts
and finally disappears.
OOCYTE DIFFERENTIATION: CENTRIOLES ON THE
MOVE
Oocyte differentiation requires a major rearrangement of the
cytoskeleton. After the last round of mitosis, the centrioles of
the cystocytes loose their anchorage to the nucleus and migrate
through the intercellular bridges towards the presumptive
oocyte (Mahowald and Strassheim, 1970). The fate of the
pericentriolar material following centriole detachment from
their nuclei is unclear. The mechanisms which drive and
control centriole congression are not known. It has been
proposed that centriole migration is mediated by the stream of
components which flow into the presumptive oocyte
(Mahowald and Strassheim, 1970). Carpenter (1994), on the
other hand, suggests that centriole migration takes place before
the start of the stream into the oocyte. Theurkauf et al. (1993)
have proposed that migrating centrioles are specifically
inactivated and slide over the microtubules nucleated by the
active centrioles of the oocyte. As they accumulate into the
oocyte, the centrioles define a large MTOC which becomes the
major microtubule organising centre of the early cyst
(Theurkauf et al., 1993 and Fig. 2). Microtubules with their
minus ends anchored to this MTOC grow into neighbouring
cells through the ring canals (Theurkauf et al., 1993; Li et al.,
1994; Clark et al., 1997). The function of the syncytial MTOC
is essential for oocyte determination. Treatment with
colchicine, which depolymerises microtubules or mutations in
Fig. 2. Schematic view of different microtubular arrays and MTOCs
in Drosophila. Microtubules are depicted brown, chromatin is blue,
centrioles are red, and pericentriolar material (PCM) is pale gray. In
the two figures on the right the outline of the cells is depicted black.
At metaphase, each pole of the embryonic mitotic spindle is
associated with a centrosome which contains two centrioles
surrounded by PCM. There is only one centriole per centrosome in
male meiosis-II spindles. Neither centrioles, nor PCM are found at
the poles of the female meiosis-I spindle. In the wing epithelial cells,
transalar microtubule bundles are organised from plaques (dark
brown) located in the inner side of the apical plasma membrane,
underneath the cuticle (thick black). There are no centrioles
associated to these plaques. Many transalar microtubules reach the
basal anchoring sites. The MTOC of the early egg chamber contains
many centrioles and organises a network of microtubules which
spans the entire cyst through the ring canals. The presence of
conventional PCM material around the centriole cluster has not been
established yet.
MTOCs in development 2701
egl or BicD, which disrupt the MTOC itself, impair oocyte
differentiation and result in cysts containing only nurse cells
(Koch and Spitzer, 1983; Schüpbach and Wieschaus, 1991;
Theurkauf et al., 1993). Centriole congression is one of the
initial events associated with the choice of the cell that will be
the oocyte, but may not be the first one. Lin and Spradling
(1995) have proposed that the association of the spectrosome
to only one of the two centrosomes during cystoblast division
could mark the cell that retains the mother centriole of that
centrosome as the future oocyte.
As oogenesis proceeds, the centriole cluster is lost, the
multicentriolar MTOC is disassembled, and microtubule
organisation within the oocyte undergoes a series of complex
changes. Firstly, microtubules are organised from the anterior
side of the oocyte cortex. Later on, they are arranged in a mesh
near the surface of the embryo (Theurkauf et al., 1992). These
microtubular arrays are involved in intercellular transport, axis
specification, and the correct localization of some determinants
at specific sites within the egg. There is no ultrastructural or
molecular data available on the mechanisms that organise
microtubules at this stages (see Theurkauf, 1994 and Knowles
and Cooley, 1994 for reviews on microtubule organization
during Drosophila oogenesis).
UNICENTRIOLAR CENTROSOMES AND MTOC
RESHAPING IN SPERMATOGENESIS
Centrosome behaviour also displays many characteristic
features during spermatogenesis. Of these we will only
mention two. The first one is the reductional segregation of the
centrioles during meiosis-II. The centrioles enter meiosis-II
without replication, to produce unicentriolar cells. The second
meiotic spindle is, therefore, built with centrosomes which
contain a single centriole (Fig. 2). This feature is unique to
males since female meiosis proceeds without centrioles. The
second characteristic feature is the transitory migration of the
centrosomes to the cell surface before meiosis (Tates, 1971).
After centrosome replication, the four centrioles migrate to the
periphery of the cell where they are found as two pairs of Vshaped structures with the distal end of each centriole
protruding from the surface of the cell. They remain at the
surface until shortly before meiosis when they migrate back
into the cell and define the spindle poles. During the two
meiotic divisions, a membranous sheath encloses the distal
ends of the centrioles. It is likely that it originates from an
invagination of the plasma membrane formed when the
centrioles were at the surface of the cell, but this remains to be
shown. The role of this membranous structure, which is also
found in other insects (Phillips, 1970), and the significance of
these movements to and from the cell surface are not known,
but is has recently become clear that at this stage a major
rearrangement of at least one PCM component takes place.
Antibodies against γ-tubulin, which reveal the centrosome as a
dot in spermatogonial cells, recognise two orthogonal rods
located near the surface of the cell in mature primary
spermatocytes. Immediately afterwards, during meiosis, the
same antibody recognises a dot-like centrosome at the poles of
the meiotic spindle and the rod-like structures are no longer
present (Wilson et al., 1997).
The preceding are just a few examples of how the
Fig. 3. Schematic view of aberrant spindles produced by mutations in
Drosophila. Microtubules are depicted brown, chromatin is blue,
centrioles are red, and pericentriolar material (PCM) is pale gray.
Mutation in ms(1)51 results in monoastral bipolar polar figures
during male meiosis-II due to failure in centrosome segregation
(Lifschytz and Meyer, 1977). The anastral pole does not contain any
centrioles, while the astral pole contains two (there is one centriole
per pole at this stage in the wild type; see Fig. 2). Anastral bipolar
spindles can be observed in embryos derived from females carrying
mutations in γTUB37C. Monopolar spindles can be observed as
‘hemi-spindles’ like the ones found in asp (Gonzalez et al., 1990)
and KLP61 (Heck et al., 1993) or as circular figures like in mgr
(Gonzalez et al., 1988) and aurora (Glover et al., 1995). The astral
pole of both, monopolars and monoastral bipolars may have a single
centrosome or more than one centrosomes which have failed to
segregate. Multipolar spindles have been observed in cells containing
multiple centrosomes like polo spermatocytes (Sunkel and Glover,
1988). In the monopolar and multipolar spindles we have also
depicted the centrioles because they are likely to be present, but it
has never been formally proven.
developmental programme requires a high degree of dynamism
from the MTOCs which are subjected to intracellular
movements, intercellular migration and re-shaping. Although,
in some cases, the functions of these changes have not been
elucidated, in others we know that they are an essential step in
the process of cell determination and differentiation.
MEMBRANE-BOUND MTOCS: A DISTANT RELATIVE
OF CENTROSOMES?
There are numerous examples of cells which loose their
centrosomes during differentiation. In these cells, microtubule
organisation takes place at sites which are not in association
with centriole-containing centrosomes (Bre et al., 1987;
Calarco-Gillam et al., 1983; Houliston et al., 1987; Szollosi et
al., 1972; Tassin et al., 1985). Drosophila wing epidermal cells,
the cells of the myotendon junction, and the cone cells of
developing ommatidia are some of these. Cone cells, which do
not possess conventional centrosomes, contain a transcellular
array of about 250 microtubule bundles associated with
2702 C. González, G. Tavosanis and C. Mollinari
plaques located at the apical and basal sides of the plasma
membrane (Mogensen et al., 1993). Wing epidermal cells also
contain microtubule bundles which span the entire length of
the cell. The minus ends of these microtubules are tightly
bound to plaques of electron-dense material located in the inner
side of the apical cell membrane and their plus ends are found
near the basal anchoring site (Tucker et al., 1986; Mogensen
et al., 1989). These trans-alar microtubule arrays are formed at
two stages during wing development (Fristrom et al., 1993;
Tucker et al., 1986; Tucker, 1992). Early microtubule bundles
are formed while the centrosome is still present, but late
bundles appear after the centrosome has been lost. Late
microtubules are mainly made of 15 protofilaments. Similar
microtuble arrays organised from membrane-associated
plaques are found in the myotendon junction of the indirect
flight muscle of Drosophila, one within the muscle cell and the
other two within the tendon cell (Reedy and Beall, 1993a,b).
The molecular composition and function of the membranebound MTOCs is still unclear. Firstly, there are contradictory
reports regarding the presence of highly conserved centrosomal
markers like γ-tubulin and pericentrin (Meads and Schoer,
1995; Rizzolo and Joshi, 1993; Mogensen et al., 1997).
Secondly, it still remains to be elucidated whether these
structures work as mere docking sites for pre-formed
microtubules as proposed by Mogensen and colleagues (1997)
or can, on their own, promote microtubule assembly.
Another instance of membrane-associated MTOCs is found
in the head of the maturing sperm. The head of Drosophila
sperm contains two major microtubular arrays: a large bundle
at the concave side of the nucleus and a smaller one which
ensheathes the convex side. Both of them are thought to be
required for sperm head shaping and elongation (Tokuyasu,
1974). The organisation of these arrays is largely unknown, but
it is clear that they originate far away from the centriole (Tates,
1971). Interestingly, in the Drosophila acentriolar cell line
1182-4, microtubule re-polymerization experiments show that
microtubule nucleation starts from the cortex of the nuclear and
plasma membranes (Debec et al., 1995). Microtubule growth
from the region surrounding the nuclear membrane has also
been reported in terminally differentiated cells of other species
(Tassin et al., 1985).
Membrane-bound MTOCs and classical centrosomes share
very little in common in terms of the presence of centrioles,
subcellular localisation, size and shape. Yet, both of them
behave as the major MTOC of the cells in which they are
found. It is, therefore, conceivable that they may contain some
common essential microtubule organising proteins together
with others which make them unique. The identification and
characterisation of these genes will be essential to understand
the function of MTOCs through development.
THE CENTROSOME AND SPINDLE ASSEMBLY
There is an open debate about the role of centrosomes in
spindle assembly. While some evidence from several systems
supports the need for centrosomes (Sluder and Rieder, 1985;
Rieder and Alexander, 1990; Zhang and Nicklas, 1995a,b)
other reports suggest that they are dispensable (Dietz, 1966;
Karsenti et al., 1984; Steffen et al., 1986; Theurkauf and
Hawley, 1992; Albertson and Thompson, 1993; Church et al.,
1986; Heald et al., 1996). The possibility that biological
differences between meiotic and mitotic cells could account for
these seemingly contradictory observations has been pointed
out by Rieder et al. (1993). In fact, the situation may even be
more complicated since male and female meiosis are
significantly different and there are many kinds of mitosis in a
developing organism. Drosophila illustrates this interpretation
since the observations regarding this issue are different
depending on the cell type and developmental stage. During
somatic cell division the role of centrosomes is largely
undisputed. The situation is not so clear during male meiosis.
Finally, there is the case of the female meiotic spindle which
is assembled without centrosomes. There is also the acentriolar
cell line 1182-4 mentioned before, which is being used to study
the role of centrosomes in spindle assembly.
SPINDLE ASSEMBLY WITHOUT CENTROSOMES
The one case in which spindle assembly occurs under natural
conditions in the absence of centrosomes is female meiosis.
The Drosophila female meiotic spindle is atypical because the
microtubules seem to originate from around the chromatin and
its poles are anastral and do not contain centrioles (Huettner,
1933; Sonnenblick, 1950; Puro and Nokkala, 1977; Hatsumi
and Endow, 1992; Theurkauf and Hawley, 1992). CP190, CP60
and γ-tubulin, which are found in the PCM of mitotic cells,
have not been detected at the poles of the female meiotic
spindle (Matthies et al., 1996; Riparbelli and Callaini, 1996;
Tavosanis et al., 1997). Thus, it seems quite clear that canonical
centrosomes of the kind found in mitotic cells are absent in the
meiotic spindle of Drosophila females. What is not so clear is
the possible role of the gene products which characterise
mitotic MTOCs in the assembly of the female meiotic spindle.
Mutation in the γTUB37C isoform of γ-tubulin, for instance,
has recently been shown to result in abnormal female meiotic
spindles (Tavosanis et al., 1997). The failure to visualise a
localised signal of γ-tubulin at the poles of these spindles
suggests that either it gets detached from microtubules before
the bipolar spindle is formed or it is evenly scattered over the
spindle. A definitive answer to the question of whether other
components of the mitotic centrosome may be involved in
building up the female meiotic spindle will be provided when
mutant alleles of those genes are identified.
Female meiosis is the only case in which the spindle is built,
under normal conditions, in the absence of centrosomes in
Drosophila. Nevertheless, certain mutant conditions can mimic
this situation. This has been observed in mature oocytes,
embryos during the syncytial blastoderm stage and
spermatocytes. In nod mutant oocytes, mini-spindles
associated to the non-exchange chromosomes which slide
away from the female meiotic spindle have been observed
(Theurkauf and Hawley, 1992). It is conceivable that the same
mechanisms that build up the female meiotic spindle organise
these minispindles. Early embryos laid by mothers which are
mutant for γTUB37C also display bipolar microtubular arrays
associated to condensed chromatin (Tavosanis et al., 1997)
(Fig. 3), but there is no evidence yet that they are functional.
The ability of embryonic systems to organise spindle-like
structures around chromatin was first reported in Xenopus eggs
where microinjected naked DNA is sufficient to organise
MTOCs in development 2703
microtubule arrays in metaphase cytoplasm in the absence of
centrosomes (Karsenti et al., 1984). Interestingly, this is not the
case in Drosophila where microinjected DNA does not
organise microtubules (Yasuda et al., 1991). Centriole-less,
spindle-like structures can also form around chromosomes
which have been pulled out of the nucleus during male meiosis
(Church et al., 1986). Unlike the cases discussed above, this
ectopic spindle can be regarded as functional in as much as the
two chromosomes of the bivalent will segregate to each pole
of the mini-spindle at the same time as their partners which
were left behind in the main spindle.
Finally, there is an interesting case of an acentriolar cell line
which is being used to study the role of centrosomes in spindle
assembly. Despite the absence of recognizable centrioles, cell
line 1182-4 proliferates, thus opening the question of how is
the spindle assembled in these cells (Debec et al., 1982). The
mitotic spindles of 1182-4 cells are very heterogeneous
including multipolar, barrel-shaped bipolar, and normal bipolar
spindles (Debec et al., 1995). The region in which the
microtubules of the spindle converge contain scattered foci of
dense filaments reminiscent of PCM (Debec et al., 1982).
These spindles contain some γ-tubulin at the poles, but this
localisation is microtubule-dependent and they do not contain
CP190. During interphase, γ-tubulin is not localised in these
cells. Debec et al. (1995) have proposed that mitotic spindle
assembly may not be carried out by the same mechanism in
wild-type and acentriolar Drosophila cells. In the wild type the
centrosomes organise spindle assembly while in the mutant, in
the absence of centrosomes, a cryptic mechanism takes over.
The efficiency of this mechanism allows the 1182-4 line to
proliferate, but cannot prevent nearly half of the mitotic figures
from being abnormal.
MUTANTS REVEAL DIFFERENT MECHANISMS OF
SPINDLE ASSEMBLY
The phenotypes of some mutants which alter the centrosome
cycle have revealed some cell-type dependent differences in the
role of centrosomes in spindle assembly. One of such mutant
phenotypes is the presence of bipolar spindles with only one
aster. These monoastral bipolar spindles resemble very closely
their wild-type counterparts, except for the absence of the aster
in one of the poles (Fig. 3). Monoastral bipolar spindles have
been observed during syncytial blastoderm divisions (D.
Glover, personal communication) and male meiosis. There are
two mutants, ms(1)RD7 and ms(1)516 that result in monastral
bipolar spindles during the first and second meiotic divisions,
respectively (Lifschytz and Hareven, 1977; Lifschytz and
Meyer, 1977). The different extent to which both meiotic
divisions are affected by these two mutants, and the fact that
neither of them has any effect in mitosis, provide a good
example of functional changes in microtubule organisation
during development. In the case of ms(1)516, segregation is
severely disrupted as most chromosomes move to the astral
pole. In this mutant, the anastral pole and the resulting daughter
cell do not contain any centrioles, while the astral pole contains
two (there is one centriole per pole at this stage in the wild
type). Therefore, the monoastral bipolar condition seems to be
due to a failure in centrosome segregation.
Interestingly, a similar failure in centrosome segregation in
other cell types results in a different phenotype. This is the case
of the serine-threonine kinases polo (Llamazares et al., 1991)
and aurora (Glover et al., 1995). Mutations in either of these
two genes impair centrosome segregation, but unlike the
previous example, monopolar instead of monoastral bipolar
spindles are formed. In this monopolar structures the
microtubules radiate from the single pole which contains the
two unsegregated centrosomes (Fig. 3). Monopolar structures
organised by either single unreplicated centrosomes or multiple
centrosomes which have failed to segregate are common to
many mitotic mutants in Drosophila (see Gonzalez et al., 1994,
for a review). In some mutants, like asp for instance,
monopolar spindles are formed in which one side of the
chromosome plate is attached to a hemi-spindle which contains
centrosomal material and the other side is attached to a bundle
of microtubules (Gonzalez et al., 1990 and Fig. 3). Judging by
the length of this bundle and the extent to which these
microtubules converge to define a pole, most of these figures
are clearly different from bipolar monoastral spindles, although
in some instances the difference may be hard to asses.
The correlation between centrosomes and spindle poles is
also substantiated by the phenotype of mutations which impair
cytokinesis. Failure of cytokinesis does not arrest the cell cycle
which proceeds with increasing amounts of unsegregated
genetic material and centrosomes. After a few mitotic cycles
without cytokinesis, cells with supernumerary centrosomes can
be observed displaying branched, multipolar spindles. Such is
the case of peanuts (Neufeld and Rubin, 1994) and pebble
(Hime and Saint, 1992). Thus, in these cases the number and
position of the centrosome dictates the shape of the mitotic
spindle so that the presence of chromosomes per se cannot
ensure the assembly of a bipolar spindle.
In summary, the only naturally occurring acentriolar spindle
which does not contain recognisable centrosomal markers in
Drosophila is found during female meiosis. Male meiotic
spindles and those of mitotic cells contain centrosomes. In
these cells, the disruption of the centrosome results in severe
alterations of spindle morphology and function. These
abnormalities appear to be different depending on the cell type
and developmental stage.
CONCLUSIONS AND FUTURE PERSPECTIVES
Microtubule organisation undergoes dramatic changes
throughout development. Different cell lineages and
developmental stages display remarkably different microtubule
arrays which in many instances are essential for the specific
functions performed by the cells in which they are found. Like
the microtubule arrays that they organise, the MTOCs of a
developing organism show a considerable degree of
morphological and functional variability. Such is the case of
the centrosomes found during early embryogenesis, those of
the meiocytes of both sexes, and the MTOCs of terminally
differentiated cells, to mention only a few examples. The study
of MTOC plasticity during development has only just started
and the questions to be answered are numerous. Firstly, we
need to further characterise the different MTOCs which occur
during Drosophila development. A particularly interesting
point is the transition between proliferating cells which contain
a typical centrosome and differentiating cells which have a
2704 C. González, G. Tavosanis and C. Mollinari
non-centrosomal MTOC. It is essential to determine the fate of
the centrosome after the last mitosis to establish whether it gets
disposed off or reconverted into the new MTOC. We also need
to determine the functional significance of the changes
undergone by MTOCs and the correlation between MTOC
specialisation and the developmental programme. Finally, we
need to identify the gene products which build and regulate the
function of these MTOCs. We expect that, on the one hand, the
essential components which provide the basic microtubule
nucleating and docking functions will be present in most
MTOCs. On the other hand, each of these MTOCs will surely
contain specific gene products which make them unique.
Characterising the common components will provide new
insights into the essentials of microtubule organisation. The
characterisation of the components which are specific to
particular cell types will allow us to unravel the relationship
between MTOCs and cell differentiation.
We are grateful to S. Reinsch, B. Lange, L. Bejarano, M. D.
Ledesma and two referees who provided many helpful comments.
Work in our laboratory is supported by the Human Capital and
Mobility programme of the European Community (CT96-005970930). G.T. and C.M. are recipients of an EMBL predoctoral
fellowship
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