T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $15.00
The Journal of Cell Biology, Vol. 177, No. 1, April 9, 2007 13–20
Centrosomes are critical microtubule (MT) nucleators and orga-
nizers in animal cells (Alberts et al., 2002). Centrioles form the
centrosome core and are surrounded by pericentriolar material
(PCM) containing MT nucleating factors like γ-tubulin (γtub;
Delattre and Gonczy, 2004). Centrosomes play key roles in
many processes, including organizing mitotic spindle poles
(Kellogg et al., 1994).
In animal cells, centrosome duplication occurs by a con-
served cycle (Alberts et al., 2002). It begins with centriole
disengagement in late mitosis (Kuriyama and Borisy, 1981),
followed by procentriole assembly along the wall of each
centriole in S phase. By G2, cells contain two mother/daughter
centriole pairs that remain in proximity until mitosis. Both cen-
triole pairs form functional centrosomes, maturing synchro-
nously before mitotic entry, by recruiting PCM and acting as
MT organizing centers (MTOCs; in contrast, there is a 10-min
delay in activating the second yeast MTOC; Shaw et al., 1997).
The centrosomes then move to opposite sides of the nucleus
to organize spindle poles and asters that position the spindle
with respect to cortical cues. The essential role of centrosomes
in animal cells was called into question by the fact that flies
lacking functional centrosomes, or lacking centrioles entirely,
live to adulthood (Megraw et al., 2001; Basto et al., 2006).
However, not all is well: these animals have defects in divisions
of larval neural stem/progenitor cells, the central brain neuro-
Adult tissue stem cells play key roles in tissue maintenance/
repair (Nystul and Spradling, 2006). In each division, the daugh-
ters differ in fate: one retains stem cell character and the other
differentiates. Drosophila melanogaster central brain NBs are a
superb model for asymmetric divisions of postembryonic tissue
stem cells (Savoian and Rieder, 2002; Siller et al., 2005). Both
embryonic and larval NBs are polarized cells exhibiting strict
division patterns crucial for their roles as stem cells. Unlike the
precise relationship between the embryonic NB division axis
and adjacent epithelium, larval central brain NBs (Fig. 1 A) do
not appear to divide with specifi c orientations relative to the
brain as a whole (Fig. 1 B). However, each NB creates a simpler
microenvironment (Fig. 1 C): the NB and its ganglion mother
cell (GMC) daughters. NBs divide asymmetrically, and the NB
daughter retains stem cell character, whereas the GMC daughter
goes on to differentiate. NBs divide according to strict local
rules; each GMC is born adjacent to the previous GMC (Fig. 1 E
and Video 1, available at http://www.jcb.org/cgi/content/full/
jcb.200612140/DC1; Akong et al., 2002), creating a GMC cap
on one side of the NB (Fig. 1, C and D).
A role for a novel centrosome cycle in asymmetric
Nasser M. Rusan1 and Mark Peifer1,2
1Department of Biology and 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
division. Earlier work showed that their mitotic spindle
orientation is established before spindle formation. We in-
vestigated the mechanism by which this occurs, revealing
a novel centrosome cycle. In interphase, the two centrioles
separate, but only one is active, retaining pericentriolar
material and forming a “dominant centrosome.” This centro-
some acts as a microtubule organizing center (MTOC) and
remains stationary, forming one pole of the future spindle.
issue stem cells play a key role in tissue mainte-
nance. Drosophila melanogaster central brain neuro-
blasts are excellent models for stem cell asymmetric
The second centriole is inactive and moves to the
opposite side of the cell before being activated as a
centrosome/MTOC. This is accompanied by asymmetric
localization of Polo kinase, a key centrosome regulator.
Disruption of centrosomes disrupts the high fi delity of
asymmetric division. We propose a two-step mechanism
to ensure faithful spindle positioning: the novel centro-
some cycle produces a single interphase MTOC, coarsely
aligning the spindle, and spindle–cortex interactions re-
fi ne this alignment.
Correspondence to Mark Peifer: firstname.lastname@example.org
Abbreviations used in this paper: GMC, ganglion mother cell; MT, microtubule;
MTOC, MT organizing center; NB, neuroblast; NEB, nuclear envelope break-
down; PCM, pericentriolar material.
The online version of this article contains supplemental material.
JCB • VOLUME 177 • NUMBER 1 • 2007 14
Although differential fate allocation is critical in stem cells,
we have much to learn about how a stereotyped division axis is
established. NBs must coordinate cortical and spindle polarity so
that neural determinants are packaged into the differentiating
daughter (Yu et al., 2006). Mutations affecting polarity or astral
MT cortical interactions result in asymmetric division defects
(Yu et al., 2006). The importance of a properly aligned spindle is
also suggested by spindle alignment defects in the absence of
centrioles (14% symmetric divisions; Basto et al., 2006) or in
mutants that lack PCM (asterless [asl] or centrosomin [cnn]) and
have few or no astral MTs (Giansanti et al., 2001; Megraw et al.,
2001). Thus, proper interactions between the spindle, astral MTs,
and cortical polarity cues help maintain a constant division axis.
Previous analyses revealed that NB spindles form at prophase
already roughly aligned with the ultimate division axis (Siller
et al., 2006) but did not defi ne how the initial axis forms.
Here, we address how this model stem cell maintains a
persistent division axis. D. melanogaster male germline stem
cells also have a persistent division axis. It was proposed that
one centrosome is cortically anchored by MT–adherens junc-
tion interactions (Yamashita et al., 2003). To test whether a simi-
lar mechanism exists in NBs, we analyzed the centrosome cycle
using 4D or 5D spinning disk confocal microscopy on brains
prepared with no physical distortion (Fig. S1 A, available at
taining NB shape to replicate normal mitosis.
Results and discussion
NBs generate a second active MTOC
during mitotic entry
By prophase, NBs contain two MTOCs that are almost fully
separated and aligned along the NB/GMC axis (Siller et al.,
2006), but analysis of fi xed NBs revealed a single MTOC posi-
tioned opposite the GMCs before mitotic entry (Ceron et al.,
2001). We thus examined MTOC behavior throughout the cell
Figure 1. NB MTOCs form asynchronously. (A) Cartoon. (B–E) NB/GMCs in central brain (B), close-up (C and E), and cartoon (D). (B and C) Phalloidin.
(E) Actin-GFP for two cell cycles; positions of successive GMCs are indicated (colored). The yellow dot represents a hypothetical MTOC. (F and G) GFP-
G147 NBs. (F) GMCs are indicated by the dotted line. Max-intensity projections are shown for entire cell. Single MTOC matures and forms MT basket
(0:00; arrow). Second MTOC appears on other side of nucleus (1:45; arrowhead) and matures (4:35). Small images show dominant MTOC (top) and sec-
ond MTOC (bottom; asterisk). (G) An end-on view of MTOC maturation, three sections of z stack. Dominant MTOC is present throughout (top, arrows).
Second MTOC appears (7:74; arrow). Dotted line indicates bottom of NB. (H) EB1-GFP. Second nucleation center appears (1:09; arrow). (I) Position where
second MTOC appears relative to dominant MTOC. Time is shown as h:min (E) and min:s (F–H). Bars, 10 μm.
A NOVEL CENTROSOME CYCLE IN NEUROBLASTS • RUSAN AND PEIFER15
cycle as an initial approach to test the hypothesis that fi xing the
position of one MTOC through successive divisions helps
ensure persistent spindle orientation. We analyzed live NBs ex-
pressing GFP-G147, an MT-associated protein (Morin et al.,
2001), revealing a striking temporal difference in MTOC activity.
During interphase, a single detectable MTOC persists opposite
the previous division site; we refer to this as the dominant
MTOC. As NBs approach mitosis, this MTOC increases activity
(matures; empirically judged by size and MT fl uorescence
intensity), forming an MT basket around the nucleus (Fig. 1, F
and G, 0:00, arrows; and Videos 2 and 3, available at http://
www.jcb.org/cgi/content/full/jcb.200612140/DC1). We refer to
this as preprophase; this stage is also seen in fi xed samples
stained for tubulin (Fig. S1 B). Soon after, sometime before the
dominant MTOC fully matures, something striking happens:
a second MTOC appears distant from the fi rst (Fig. 1 F, 1:45,
arrowheads; and Video 2). We refer to this as the second MTOC
and this stage as prophase onset. The second MTOC increases
activity, maturing ?10 min before nuclear envelope breakdown
(NEB; Fig. 1 F, 1:45–13:24). Using 4D imaging, we excluded
the possibility that the second MTOC was present earlier in
another focal plane. To further assess this, we imaged forming
spindles end on (Fig. 1 G and Video 3). It is clear that the second
MTOC did not emerge from the dominant MTOC (Fig. 1 G,
top) or travel around the nucleus (Fig. 1 G, middle). Instead, the
second MTOC appeared roughly opposite the dominant MTOC
(Fig. 1 G, bottom, arrowheads; 132 ± 38° from the dominant
one, using the centroid of the nucleus as a fi xed reference; n = 30;
Fig. 1 I, prophase). MTOC separation began immediately, and
by NEB, they were 146 ± 20° apart (n = 18, Fig. 1 I; this is
slightly less than seen by Siller et al., 2005 [171°], likely be-
cause of different measurement methods). Thus, NBs form two
distinct MTOCs: an MTOC persisting from the previous divi-
sion and another only activated at mitotic entry.
A novel centrosome cycle in NBs
This distant activation of the second MTOC raised questions
about the centrosome cycle. One possibility is that NBs have
two MT nucleating centrosomes, but only one can retain MTs
and act as an MTOC during interphase, whereas the second
acquires MT retention ability during mitotic entry, explaining
the second MTOC’s sudden appearance. There is precedent for
this: mouse L929 cells have two γtub-bearing centrosomes that
can nucleate MTs, but only one contains Ninein and can retain
MTs to form an MTOC (Piel et al., 2000).
To test this hypothesis in NBs, we used EB1-GFP. This
binds growing MT plus ends and reliably identifi es MT nucle-
ation sites (Mimori-Kiyosue et al., 2000; Piehl et al., 2004).
Only one nucleation site was present in interphase and prepro-
phase (Fig. 1 H, arrows; 0:00; z series not depicted), and a new
nucleation site appeared distant from the fi rst (Fig. 1 H, arrow-
heads), consistent with spatially and temporally distinct second
MTOC activation. Thus, NBs regulate MT nucleation and not
just MT retention.
To examine how the new nucleation center forms, we
imaged centrosomes using a PCM protein, GFP-Cnn (Megraw
et al., 2002). NBs contain a single detectable centrosome during
interphase (Fig. 2 A, 0:51–2:36). When NBs reenter mitosis,
a second Cnn-positive centrosome appears distant from the
fi rst (Fig. 2 A, 2:36, blue arrow), mimicking activation of the
second MTOC. To verify that these occur simultaneously, we
imaged NBs expressing mCherry-Tubulin (chTub) and GFP-
Cnn (Fig. 2 B and Fig. S2 B, available at http://www.jcb.org/
cgi/content/full/jcb.200612140/DC1). This revealed perfect
temporal and spatial correlation between the appearance of
the second centrosome and activation of the second MTOC
(Fig. 2 B, arrowheads). We never saw physical separation of two
centro somes/MTOCs (n > 60). To our knowledge, this is the fi rst
example of asynchronous and physically distant centrosome
maturation, suggesting that NBs use a novel centrosome cycle.
Higher temporal/spatial resolution imaging revealed that
two GFP-Cnn spots separate during mitotic exit (Fig. 2 B,
inset; and Video 5, available at http://www.jcb.org/cgi/content/
full/jcb.200612140/DC1). One GFP-Cnn spot persists as the
NB interphase centrosome, forming the dominant MTOC,
whereas the other spot disappears. The persistent Cnn spot
(centrosome) remains relatively stationary in interphase (see
The dominant centrosome predicts spindle alignment), consistent
Figure 2. Differential maturation of NB centro-
somes. (A) GFP-Cnn for one cell cycle. Dom-
inant centrosome is indicated by the red
arrows. PCM is reduced during mitotic exit
and accumulates in preprophase. GMC cen-
trosome completely loses PCM (green arrow-
heads). Second centrosome appears distant
from dominant centrosome (blue arrowheads).
The asterisk highlights the appearance of
the second centrosome. (B) GFP-Cnn, chTub.
Arrows indicate dominant centrosome. Arrow-
heads indicate second centrosome. The inset
shows GFP-Cnn, PCM splitting. Time is shown
as h:min. Bars, 10 μm.
JCB • VOLUME 177 • NUMBER 1 • 2007 16
with the hypothesis that coarse spindle alignment begins in
interphase by anchoring the dominant centrosome (Fig. 2 A,
0:00–2:48, red arrows).
The NB daughter cells differ
in PCM retention
We also examined centrosome fate in the two daughters (new
NB and GMC). They differ dramatically in PCM retention, in
contrast to mammalian cells, where both daughters’ centrosomes
retain PCM. The GMC centrosome sheds all PCM (Fig. 2 A,
0:27–0:51, green arrowheads; centrioles remain [see Asymmetric
centrosome regulation]; GMCs regain PCM when reentering
mitosis, Ceron et al., 2001). The new NB centrosome (that be-
comes the dominant MTOC) retains PCM throughout interphase
(Fig. 2 A, 0:27–0:51, red arrows) and further accumulates PCM
during the next mitosis (Fig. 2 A, 1:39–2:36, red arrows and
Fig. S2 A). The complete shedding of PCM in GMCs appears to
be the normal behavior of interphase centrosomes in most fl y
cells (Cottam et al., 2006; Rogers, G., personal communication),
whereas in syncytial early embryos, both daughters retain PCM
foci through the cell cycle. In contrast to both cell types, the NB
daughters exhibit differential PCM retention.
Our data suggest that NBs have a novel centrosome cycle
in which the second centrosome matures distant from the domi-
nant centrosome/MTOC. One hypothesis to explain this would
be the distal positioning of a differentially regulated centriole
that is blocked from recruiting PCM in interphase and thus
cannot form an MTOC until “activated” during mitosis. If this
centriole is always inherited by the GMC, it might also explain
complete PCM loss as GMCs exit mitosis.
Differential centriole movement
We examined centrioles live to test this hypothesis, using the
centriole marker GFP-PACT (Martinez-Campos et al., 2004)
and Histone-GFP (Fig. 3 A). Mother/daughter centrioles dis-
engaged in late telophase (Fig. 3 A, 0:24; and Fig. S1 C), as in
mammalian cells and fl y embryos (Kuriyama and Borisy, 1981;
Piel et al., 2000). Thus, two NB centrioles are present through-
out interphase despite the presence of only one MTOC.
The two centrioles then exhibit different behaviors. One
remains fairly stationary (Fig. 3 A, arrows), whereas the second
moves to roughly the other side of the nucleus (arrowheads).
Disengagement perfectly correlates with separation of Cnn
spots (Fig. 2 B), suggesting that the stationary centriole retains
PCM to form the dominant MTOC and the mobile centriole
completely sheds PCM. To test this, we imaged NBs expres-
sing chTub and GFP-PACT (Fig. 3 B and Video 6, available at
stationary centriole retained MTs (Fig. 3 B, arrows), whereas
the mobile centriole did not (arrowheads). Upon reentering mito sis,
the mobile centriole regained nucleation activity, forming the
This suggests that full separation of the MTOCs that or-
ganize the spindle is biphasic. It begins in interphase, when
Figure 3. NB centrioles differ in behavior and
regulation. NBs are outlined in A, B, E, and F.
Imaged proteins are indicated on each fi gure.
Images are displayed in inverse contrast.
(A) Centrioles separate (each indicated by an
arrow or arrowhead; 0:24; insets highlight
the separation). One is relatively stationary
(arrows), and the second moves to other side of
nucleus (arrowheads). (A’) The diagram shows
the path of centrioles in A. (B) Centrioles split
in late telophase; one remains stationary with
associated MTs (arrows), and the other loses
MTs and moves around the nucleus (arrow-
heads). (C and D) Fixed NBs. DPLP and MTs
(C) or γtub (D) are shown. (C) Preprophase
NB. One centriole is at the center of MT aster
(arrows), and the other has no associated MTs
(arrowheads). Asterisks show centrioles in ad-
jacent cells that appear to be in NB by max-
intensity projection. (D) Only the centriole distant
from GMC cap has γtub (arrows). GMC centri-
oles lack γtub (yellow arrowheads). (E and E’)
Mitotic entry: preprophase dominant centriole
has Polo (arrows), and the second does not
(arrowheads). (0:04) Prophase; second centri-
ole begins to acquire Polo (arrowheads). After
NEB, Polo moves to kinetochores (0:21–0:35;
red arrowheads). (F) Mitotic exit: NB retains
Polo on dominant centriole from telophase into
interphase (0:00–1:36; black arrows). Polo is
also at midbody in telophase (blue arrows).
Time is shown as h:min. Bars, 10 μm.
A NOVEL CENTROSOME CYCLE IN NEUROBLASTS • RUSAN AND PEIFER17
one centriole retains PCM, remains stationary, and forms the
dominant MTOC, whereas the second centriole sheds PCM
and becomes mobile. Movement of the second centriole away
from the dominant MTOC in interphase accounts for ?70%
(132/180°; Fig. 1 I) of the separation needed to form a spindle.
Mecha nisms of transporting the mobile centriole remain to be
identifi ed, but it is nonrandom, as in 26/30 NBs, the second
MTOC emerged ≥90° from the dominant MTOC (Fig. 1 I).
After the second MTOC is activated, the two separate the last
30%, most likely via MT sliding forces. This might explain
defects in lissencephaly1 mutants, where MTOCs are only sep-
arated by 124° at NEB (Siller et al., 2005). Perhaps interphase
centriole movement is normal, but MT-based MTOC separation
Asymmetric centrosome regulation
These data suggest that NBs differentially regulate the activity
of their two centrioles within the same cytoplasm. Interestingly,
a similar observation was made in clam eggs, which have three
centrosomes just after fertilization. The sperm centrosome is
functionally inactivated, whereas female centrosomes organize
the meiotic spindle (Wu and Palazzo, 1999).
We next examined NB centrosome regulation. In prepro-
phase, one centriole (marked by anti-DPLP; Fig. S1 C) formed
the dominant MTOC (Fig. 3 C, arrows), whereas the second
centriole had no associated MTs and was randomly positioned
(Fig. 3 C, arrowheads), confi rming our live-cell data. We thus
examined whether γtub is recruited asymmetrically. Fixed pre-
prophase NBs had two centrioles; only that opposite the GMCs
accumulated γtub (Fig. 3 D; neither GMC centriole carried γtub
[yellow arrowheads], consistent with complete Cnn loss in
interphase GMCs). Further, both γtub and Cnn are absent from
the NB centriole nearest the GMCs in interphase/preprophase
(Fig. S2 C).
Polo kinase promotes centrosome maturation by pro-
moting γtub recruitment during mitotic entry (Glover, 2005).
Differences in Polo localization/activity might underlie differ-
ences in timing of NB centrosome maturation. We examined NBs
expressing Polo-GFP and the centriole marker mCherry-DSAS-6
(Dammermann et al., 2004). Only the centriole pair that forms
the dominant centrosome was Polo-GFP positive during prepro-
phase (Fig. 3 E, arrows). Polo-GFP accumulated on the mobile
centriole pair as the NB entered mitosis (Fig. 3, E and E′, arrow-
heads; and Video 7, available at http://www.jcb.org/cgi/content/
full/jcb.200612140/DC1), increased on both centriole pairs
through prophase, and moved on to kinetochores (Moutinho-
Santos et al., 1999). When we imaged Polo-GFP in cells exiting
mitosis, we could see it retained at low levels on the dominant
centrosome (Fig. 3 F and Fig. S2, D–G). In the future, it will be
interesting to examine the localization of Aurora A, another
Unlike the distal appendages of mammalian mother cen-
trioles, fl y mother and daughter centrioles have no known ultra-
structural (Callaini and Riparbelli, 1990) or molecular differences.
Figure 4. Two phases of spindle alignment. (A) Cartoon showing measured angles. (B) Centrosome location relative to anaphase division axis onset.
Green circles indicate the dominant centrosome, and blue circles indicate the second centrosome. In 1/25 NBs, the dominant centrosome (dark green) was
on the GMC side of the nucleus at prophase; its second centrosome is shown in dark blue. Measurements used Cnn (blue; interphase and prophase) or
MTs (pink). (C) Sample video stills. Yellow lines indicate anaphase-onset axis. (D) Cartoon showing centrosome/centriole cycles. Time is shown as h:min.
Bar, 10 μm.
JCB • VOLUME 177 • NUMBER 1 • 2007 18
Our data suggest that differences exist. It is unlikely that this
differential regulation is a result of location, as both centrioles
are initially adjacent after disengagement. The differences may
be due to centriole age or procentriole maturation state.
The dominant centrosome predicts
The NB spindle is largely aligned by NEB (Siller et al., 2006).
Based on our data, we tested the hypothesis that the dominant
centrosome helps defi ne one spindle pole before prophase. We
calculated the angle between the dominant centrosome/MTOC
axis (Fig. 4 A, top) and the anaphase axis (bottom), using the
nuclear centroid as a fi xed reference. This revealed two phases
in defi ning the future spindle axis. Through prophase onset, the
dominant centrosome remains fairly stationary roughly oppo-
site the GMCs (Fig. 4 B, coarse alignment), agreeing with fi xed
images (Ceron et al., 2001), whereas the second centriole moves
to a distal position (to within 46 ± 33° [n = 25] of the anaphase
axis; Fig. 4 B, prophase). This is consistent with our hypothesis.
The dominant centrosome may be immobilized by aster–cortex
interactions or by absence of an active displacement mechanism.
In the second phase, alignment is refi ned in prophase and pro-
metaphase (the angle between the NB centrosome and anaphase
axes decreases from 31 ± 29° to 15 ± 12°; n = 15), as shown
by Siller et al. (2006).
To further test whether anchoring the dominant centro-
some helps roughly align the spindle, we imaged asl mutant
NBs live. They lack functional centrosomes (Giansanti et al.,
2001; Fig. S3 A, available at http://www.jcb.org/cgi/content/
full/jcb.200612140/DC1) and astral MTs. Mutant NBs lack a
dominant interphase centrosome, allowing us to assess its role
in spindle orientation and asymmetric cell division. Live im-
aging revealed robust chromatin-mediated MT nucleation and
spindle assembly producing fairly normal spindles (Fig. 5 A
and Video 9). Spindle poles emerge from a disorganized MT
array near the chromosomes that focuses as the spindle length-
ened. Spindles do not rotate during formation, always forming
along the initial pole separation axis, but do rotate during meta-
phase (23 ± 15°; n = 11), suggesting that rotation can occur
without astral MTs or that asl mutants have a reduced astral
array suffi cient for rotation (Fig. 5 A). Surprisingly, consecutive
divisions in asl mutants usually produce adjacent or near-
adjacent daughters (n = 5/5; Fig. 5 B and Video 10), as in wild
type (Fig. 1 E). In a few cases, however, spindles form parallel
to the GMC cap and, presumably, the polarity axis (2/13;
?15%); these NBs divide symmetrically (Fig. 5 C). This sug-
gests that the second phase of spindle alignment can occur
without a dominant centrosome and can rescue misalignment,
as long as it is not too extreme, but occasional atypical sym-
metric divisions occur. This results in defective brain anatomy,
Figure 5. Functional centrosomes ensure high-fi delity division asymmetry. (A–C) GFP-G147 in asl. (A) Chromosome-induced spindle assembly (0:01–0:09).
Initial spindle alignment is absent, but refi nement occurs (0:09–0:12). (B) Two rounds of mitosis. GMCs born near one another. Arrowheads indicate fi rst
daughter. (C) Example where initial spindle alignment was far off NB-GMC axis, with resulting symmetric division. (D) Mechanistic model of the importance
of dominant interphase MTOC. (E) Hypothetical case: centrosomes matured synchronously as in canonical cycle. (F) Division with no centrosomes. Pound
sign indicates that number is from fi xed analysis of telophase NBs (Giansanti et al., 2001). Time is shown as h:min. Bars, 10 μm.
A NOVEL CENTROSOME CYCLE IN NEUROBLASTS • RUSAN AND PEIFER19
with ectopic paired, smaller NBs, presumably progeny of sym-
metric divisions (Fig. S3 B).
A novel centrosome cycle helps ensure
fi delity of spindle position
Our data reveal two new aspects of asymmetric division in this
stem cell model. First, cells can adjust the canonical centro-
some cycle to allow novel cell behaviors, as was observed
during clam meiosis (Wu and Palazzo, 1999). Central brain
NBs also alter this cycle: rather than both centrosomes maturing
in synchrony and proximity (Fig. 5 E), the two centriole
pairs are differentially regulated, maturing asynchronously
and distant from one another (Fig. 5 D). One retains MT nu-
cleating activity throughout the cell cycle, forming the domi-
nant MTOC during interphase, whereas the second is initially
inactive, only forming a functional centrosome and nucleating
MTs at mitotic entry. One speculative possibility is that these
are mother and daughter centrioles and that one is preferen-
tially retained in the stem cell, a hypothesis that will now be
tested. It is also of interest to ask whether other stem cells use
Second, our data suggest that this novel centrosome cycle
helps ensure high-fi delity spindle positioning and thus asym-
metric division (Fig. 5 D). We propose a model in which NB
mitotic spindles are aligned in two phases to ensure that GMC
daughters are born next to the previous GMC. Rough alignment
is achieved by confi ning the dominant MTOC to a relatively
fi xed position from the previous division and moving the sec-
ond centriole to the other side of the cell. As spindles form,
a second process refi nes this initial alignment. In asl mutants,
without centrosomes, the fi rst mechanism is inactive, but the
second mechanism can align the spindle unless initial alignment
is wildly off axis (Fig. 5 F). In mud mutants, centriole separa-
tion must occur normally, as prophase MTOCs are nearly fully
separated, but alignment of spindle poles to cortical polarity
cues is defective (Siller et al., 2006). The normal two-step
process is a robust mechanism ensuring successful asymmetric
divisions and reproducible brain anatomy.
Materials and methods
y w fl ies were the wild-type controls for all immunostained samples. For
live-cell imaging, we used the following strains: UAS-actin-GFP (Jacinto
et al., 2000), UAS-GFP-Cnn1 (Megraw et al., 2002), GFP-G147 (GFP-
tagged MT-associated protein; Morin et al., 2001), UAS-EB1-GFP (a gift
from S. Rogers [University of North Carolina at Chapel Hill, Chapel Hill,
NC] and B. Eaton [University of Texas at San Antonio, San Antonio, TX]),
GFP-PACT (Martinez-Campos et al., 2004), and Polo-GFP (Moutinho-
Santos et al., 1999). We generated transgenic fl ies of the genotype UAS-
mCherry-α-tubulin and mCherry-SAS-6 by using a standard P-element
transformation (Rubin and Spradling, 1982). mCherry-α-tubulin (human
tubulin) was PCR amplifi ed from an unknown expression vector (a gift from
A. Straight, Stanford University, Stanford, CA) and cloned into the pUASg
vector. mCherry-SAS-6 (generated by G. Rogers, University of North Caro-
lina at Chapel Hill) is expressed under its endogenous promoter and was
cloned into the pCaSpeR4 vector. All UAS promoters were driven by Gal4-
1407 (Bloomington Drosophila Stock Center). For fi xed samples of asl
mutants, we identifi ed homozygous asl2 larvae by selecting against the Tubby
marker on the TM6 Balancer (Giansanti et al., 2001). For live imaging of
MTs in the asl background, we generated recombinants of the genotype
Live-cell imaging of NBs in the intact larval brain
Crawling third instar larvae were dissected in Schneider’s Drosophila
Medium (Invitrogen) with 10% FCS. The entire brain was explanted and
placed anterior side down (ventral nerve cord upward) in our imaging
chamber (Fig. S1). Brains were allowed to settle in the center of a pool of
media in a glass-bottomed dish (MatTek). The media was surrounded by
Halocarbon oil 700, which supported a glass coverslip used to seal the
chamber. Samples were imaged using a Yokogawa spinning disk confocal
(PerkinElmer) mounted on a microscope (Eclipse TE300; Nikon). It is
equipped with an interline cooled charge-coupled device camera (ORCA-
ER; Hamamatsu), a z-focus motor (Prior Scientifi c), an excitation and an
emission wheel controlled by the Lambda 10-2 controller (Sutter Instrument)
and emission fi lters from Semrock. Objectives used were 100× 1.4 NA,
60× 1.4 NA, and 40× oil 1.3 NA. 4D and 5D (x, y, z, time, wavelength)
video sequences were collected using the multidimensional acquisition
add-on in MetaMorph (Molecular Devices).
Brains of y w and asl2/asl2 fl ies were fi xed in 9% formaldehyde or 4%
paraformaldehyde in PTA (PBS + 0.1% Tween 20 + 0.2 g/l sodium azide)
for 15 min, blocked in 1% normal goat serum for 3 h, and stained in a
microcentrifuge tube in primary antibody and 1% normal goat serum in PTA
overnight at 4°C. Brains were washed and incubated in secondary anti-
bodies for 2 h at room temperature. The following antibodies were used:
E7 mouse anti–α-tubulin (1:250; Developmental Studies Hybridoma Bank),
rabbit anti-DPLP (1:1,000; Martinez-Campos et al., 2004), mouse GTU-88
anti–γ-tubulin (1:500; Sigma-Aldrich), and rabbit anti-GFP (1:750; ab290
[Abcam]). Secondary antibodies were Alexa 488 and 546 (Invitrogen)
and were used at a fi nal concentration of 1:500.
Measuring angles between the centrosome and the anaphase-onset
For each selected time point, the (x, y, z) coordinates of the centrosome
was recorded. We also recorded the coordinates for the point of origin at
each time point, which we designated as center of the nucleus from inter-
phase to NEB, the center of the chromosomal mass at initial metaphase,
and half the distance between the slightly separated sister chromatids at
anaphase onset. At each time point, the origin was normalized to (0, 0, 0)
and the centrosome coordinates were adjusted accordingly. This method
eliminated the effects of x, y, z stage/microscope drift. The following
equation was used to measure the angle between the two defi ned vectors (x1,
y1, z1) and (x2, y2, z2): Dot Product = (x1 × x2) + (y1 × y2) + (z1 ×
z2) = L1 × L2 × cos(Θ), where L, length of vector, equals the square root
of (x2 + y2 + z2) and Θ is the angle between the two vectors. Note that all
the measured angles are in relation to the anaphase-onset vector, which
was always designated as the (x2, y2, z2) vector.
For the interphase time points, we used GFP-Cnn (Fig. 4 B, blue),
because the interphase centrosome could not always be identifi ed with
high confi dence using an MT marker. We used GFP-G147 to stage cells at
prophase (appearance of second MTOC), NEB (fl ood of fl uorescence into
the nucleus), initial metaphase (judged by spindle shape), and anaphase
onset (kinetochore MT shortening; Fig. 4 B, pink).
Online supplemental material
Video 1 shows wild-type NBs expressing actin-GFP through two rounds
of mitosis. Video 2 presents a side view of wild-type GFP-G147–expressing
NBs during mitotic entry. Video 3 gives an end-on view of wild-type GFP-
G147–expressing NBs during mitotic entry. Video 4 shows wild-type
NBs expressing GFP-Cnn through an entire cell cycle. Video 5 shows
wild-type NB expressing chTub and GFP-Cnn, showing PCM splitting
during mitotic exit. Video 6 presents wild-type NB expressing chTub and
GFP-PACT. Video 7 shows wild-type NB expressing mCherry-SAS-6 and
Polo-GFP during mitotic entry. Video 8 shows wild-type GFP-G147–
expressing NBs during mitotic entry and through the end of telophase.
Video 9 shows asl2,G147 mutant NBs during spindle assembly and through
mitosis. Video 10 shows asl2,G147 mutant NBs through two rounds of
mitosis. Fig. S1 provides a sample preparation and MT distribution in
NBs. Fig. S2 shows CNN and Polo behavior throughout the cell cycle.
Fig. S3 shows that asl mutant brains contain supernumerary central
brain NBs. Online supplemental material is available at http://www.jcb
We thank C. Doe, T. Kaufman, P. Martin, C. Sunkel, M. Gatti, and J. Raff for
reagents; S. Rogers and B. Eaton for the unpublished EB1-GFP fl y stock;
and A. Khodjakov, T. Salmon, K. Bloom, S. Rogers, and G. Rogers for help-
JCB • VOLUME 177 • NUMBER 1 • 2007 20 Download full-text
N.M. Rusan is supported by American Cancer Society grant PF-06-
108-CCG. This work was supported by National Institutes of Health grant
Submitted: 22 December 2006
Accepted: 5 March 2007
Note added in proof. While this work was in review, two relevant papers
were published. Rebollo et al. (Rebollo, E., P. Sampaio, J. Januschke,
S. Llamazares, H. Varmark, and C. Gonzalez. 2007. Dev. Cell. 12:467–474)
also investigated centrosomes in Drosophila neuroblasts, and Yamashita et al.
(Yamashita, Y.M., A.P. Mahowald, J.R. Perlin, and M.T. Fuller. 2007. Science.
315:518–521) studied centrosomes in another Drosophila stem cell model,
the male germline stem cells.
Akong, K., B.M. McCartney, and M. Peifer. 2002. Drosophila APC2 and APC1
have overlapping roles in the larval brain despite their distinct intra-
cellular localizations. Dev. Biol. 250:71–90.
Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2002.
Molecular biology of the cell. Garland Science, New York. 1616 pp.
Basto, R., J. Lau, T. Vinogradova, A. Gardiol, C.G. Woods, A. Khodjakov, and
J.W. Raff. 2006. Flies without centrioles. Cell. 125:1375–1386.
Callaini, G., and M.G. Riparbelli. 1990. Centriole and centrosome cycle in the
early Drosophila embryo. J. Cell Sci. 97:539–543.
Ceron, J., C. Gonzalez, and F.J. Tejedor. 2001. Patterns of cell division and ex-
pression of asymmetric cell fate determinants in postembryonic neuro-
blast lineages of Drosophila. Dev. Biol. 230:125–138.
Cottam, D.M., J.B. Tucker, M.M. Rogers-Bald, J.B. Mackie, J. Macintyre, J.A.
Scarborough, H. Ohkura, and M.J. Milner. 2006. Non-centrosomal
microtubule-organising centres in cold-treated cultured Drosophila cells.
Cell Motil. Cytoskeleton. 63:88–100.
Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and
K. Oegema. 2004. Centriole assembly requires both centriolar and peri-
centriolar material proteins. Dev. Cell. 7:815–829.
Delattre, M., and P. Gonczy. 2004. The arithmetic of centrosome biogenesis.
J. Cell Sci. 117:1619–1630.
Giansanti, M.G., M. Gatti, and S. Bonaccorsi. 2001. The role of centrosomes and
astral microtubules during asymmetric division of Drosophila neuroblasts.
Glover, D.M. 2005. Polo kinase and progression through M phase in Drosophila:
a perspective from the spindle poles. Oncogene. 24:230–237.
Jacinto, A., W. Wood, T. Balayo, M. Turmaine, A. Martinez-Arias, and P. Martin.
2000. Dynamic actin-based epithelial adhesion and cell matching during
Drosophila dorsal closure. Curr. Biol. 10:1420–1426.
Kellogg, D.R., M. Moritz, and B.M. Alberts. 1994. The centrosome and cellular
organization. Annu. Rev. Biochem. 63:639–674.
Kuriyama, R., and G.G. Borisy. 1981. Centriole cycle in Chinese hamster ovary
cells as determined by whole-mount electron microscopy. J. Cell Biol.
Martinez-Campos, M., R. Basto, J. Baker, M. Kernan, and J.W. Raff. 2004. The
Drosophila pericentrin-like protein is essential for cilia/fl agella function,
but appears to be dispensable for mitosis. J. Cell Biol. 165:673–683.
Megraw, T.L., L.R. Kao, and T.C. Kaufman. 2001. Zygotic development without
functional mitotic centrosomes. Curr. Biol. 11:116–120.
Megraw, T.L., S. Kilaru, F.R. Turner, and T.C. Kaufman. 2002. The centrosome is
a dynamic structure that ejects PCM fl ares. J. Cell Sci. 115:4707–4718.
Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. The dynamic behavior of
the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol.
Morin, X., R. Daneman, M. Zavortink, and W. Chia. 2001. A protein trap strategy
to detect GFP-tagged proteins expressed from their endogenous loci in
Drosophila. Proc. Natl. Acad. Sci. USA. 98:15050–15055.
Moutinho-Santos, T., P. Sampaio, I. Amorim, M. Costa, and C.E. Sunkel. 1999.
In vivo localisation of the mitotic POLO kinase shows a highly dynamic
association with the mitotic apparatus during early embryogenesis in
Drosophila. Biol. Cell. 91:585–596.
Nystul, T.G., and A.C. Spradling. 2006. Breaking out of the mold: diversity within
adult stem cells and their niches. Curr. Opin. Genet. Dev. 16:463–468.
Piehl, M., U.S. Tulu, P. Wadsworth, and L. Cassimeris. 2004. Centrosome
maturation: measurement of microtubule nucleation throughout the
cell cycle by using GFP-tagged EB1. Proc. Natl. Acad. Sci. USA.
Piel, M., P. Meyer, A. Khodjakov, C.L. Rieder, and M. Bornens. 2000. The re-
spective contributions of the mother and daughter centrioles to centro-
some activity and behavior in vertebrate cells. J. Cell Biol. 149:317–330.
Rubin, G.M., and A.C. Spradling. 1982. Genetic transformation of Drosophila
with transposable element vectors. Science. 218:348–353.
Savoian, M.S., and C.L. Rieder. 2002. Mitosis in primary cultures of Drosophila
melanogaster larval neuroblasts. J. Cell Sci. 115:3061–3072.
Shaw, S.L., E. Yeh, P. Maddox, E.D. Salmon, and K. Bloom. 1997. Astral
microtubule dynamics in yeast: a microtubule-based searching mechanism
for spindle orientation and nuclear migration into the bud. J. Cell Biol.
Siller, K.H., M. Serr, R. Steward, T.S. Hays, and C.Q. Doe. 2005. Live imag-
ing of Drosophila brain neuroblasts reveals a role for Lis1/dynactin
in spindle assembly and mitotic checkpoint control. Mol. Biol. Cell.
Siller, K.H., C. Cabernard, and C.Q. Doe. 2006. The NuMA-related Mud protein
binds Pins and regulates spindle orientation in Drosophila neuroblasts.
Nat. Cell Biol. 8:594–600.
Wu, X., and R.E. Palazzo. 1999. Differential regulation of maternal vs. paternal
centrosomes. Proc. Natl. Acad. Sci. USA. 96:1397–1402.
Yamashita, Y.M., D.L. Jones, and M.T. Fuller. 2003. Orientation of asymmetric
stem cell division by the APC tumor suppressor and centrosome. Science.
Yu, F., C.T. Kuo, and Y.N. Jan. 2006. Drosophila neuroblast asymmetric cell
division: recent advances and implications for stem cell biology. Neuron.