doi: 10.1242/10.1242/jcs.00134
The centrosome is a dynamic structure that ejects PCM flares
Timothy L. Megraw1,2,*,
Sandhya Kilaru1,2,
F. Rudolf Turner1 and
Thomas C. Kaufman1,2,
1 Department of Biology, Indiana University, Bloomington, IN 47405, USA
2 Howard Hughes Medical Institute, Indiana University, Bloomington, IN 47405,
USA
* Present address: Cecil H. and Ida Green Center for Reproductive Biology
Sciences and Dept of Pharmacology, University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9051, USA
View larger version (38K):
[in a new window]
|
Fig. 4. Flare activity changes through the cleavage cycle. A GFP-Cnn embryo was
injected with Rhodamine-tubulin to reveal the dynamics of centrosomes and
microtubules simultaneously in living embryos. This analysis shows that flares
appear throughout the division cycle in the syncytial embryo, with flaring
occurring most actively at telophase and interphase
(Table 1). The time code in
seconds appears in the lower right corner of each frame. See also Movie 3 at
jcs.biologists.org/supplemental.
|
|
View larger version (145K):
[in a new window]
|
Fig. 1. Centrosomin localizes to centrosomes and to extracentrosomal particles. The
localization of Cnn in early syncytial embryos was determined by
immunostaining with antibodies directed against Cnn. The image of the specimen
in A was acquired with a low signal gain, showing Cnn localization at the
centrosome to have a doughnut-like appearance. In contrast to A, the specimen
in B was acquired at a high signal gain, showing the presence of
Cnn-containing extracentrosomal particles around the centrosomes. (C) An
electron micrograph of an embryonic centrosome at interphase of cycle 14 that
has been immunogold labeled for Cnn. The image shows that Cnn is a component
of the electron-dense pericentriolar material, the tentacle-like projections
of which are enriched in Cnn. The Cnn signal appears as black dots, some of
which are indicated with white arrows, that are more concentrated within the
PCM. See also Movie 1, available at
jcs.biologists.org/supplemental.
|
|
View larger version (23K):
[in a new window]
|
Fig. 2. GFP-Centrosomin localizes to centrosomes. GFP-Centrosomin fusion protein
(GFP-Cnn) was expressed in the early embryo. This expression rescues the
maternal effect lethal phenotype associated with cnn mutants and is
therefore biologically active. These representative still images show GFP-Cnn
localized to centrosomes (arrow in A), weakly to the spindle microtubules
(arrowhead in A) and to punctate particles throughout the embryo. The stills
show the first cycle in anaphase (A), late anaphase (B), telophase, when the
centrosomes duplicate (C), prophase (D) and metaphase of the next cycle (E).
The nuclei can be seen as the dark areas in D that are devoid of GFP-Cnn
signal. See also Movie 1 at
jcs.biologists.org/supplemental.
|
|
View larger version (36K):
[in a new window]
|
Fig. 3. Cnn has a dynamic relationship with the centrosome. Emerging as `flares',
GFP-Cnn particles move back and forth from the centrosome. Shown are 12
time-lapse images of a centrosome dividing at telophase. The time code in
seconds is shown at the lower left corner of each frame. The arrows indicate
the direction of flare particle movement. See also Movie 2 at
jcs.biologists.org/supplemental.
|
|
View larger version (108K):
[in a new window]
|
Fig. 5. Cnn and D-TACC colocalize at flare particles. Shown is a wild-type (OreR)
interphase syncytial stage embryo double immunostained for D-TACC (green) and
Cnn (red). DNA, stained with TOTO-3, is shown in blue. Yellow arrows indicate
several flare particles in the D-TACC, Cnn and the merged images. Not all
Cnn-immunoreactive particles contained D-TACC and vice versa. In the merged
image the D-TACC signal at the centrosome appears to extend further from the
center than does Cnn, but this is an inaccurate representation of centrosome
structure and is due to an higher gain placed on the D-TACC signal at data
collection, which was necessary to bring the flare particle signal to a
similar level to that achieved with Cnn. See also Movie 4 at
jcs.biologists.org/supplemental.
|
|
View larger version (82K):
[in a new window]
|
Fig. 6. Flares are dependent on microtubules. (A) GFP-Cnn embryos were injected
with the microtubule-destabilizing drug colchicine to inhibit the formation of
microtubules. Shown is a single still of the movie. Flare particles surround
the centrosome but no longer move back and forth as they do in the untreated
embryos. Incipient flares appear to emerge from the centrosome but do not bud
off as flare particles. (B) Flares are associated with microtubules. Shown are
images of embryos stained for Cnn (red), microtubules (green) and DNA (blue).
Flare particles are often associated with microtubules (arrows in B),
appearing at the end (solid arrow) of a microtubule (arrowhead) or projecting
out like a tentacle along microtubules (arrow). The image in B was
deconvoluted to resolve the Cnn and microtubule signals better. See also Movie
4 at
jcs.biologists.org/supplemental.
|
|
View larger version (169K):
[in a new window]
|
Fig. 7. Flare movement is dependent on microtubule dynamics rather than on
microtubule-based motors. Live embryos were treated with paclitaxel (Taxol) to
stabilize microtubules. GFP-tubulin embryos treated with Taxol (B) exhibit
intense spindle microtubules, bright asters and have an arrested cycle as
compared to mock control GFP-tubulin embryos (A). Flare activity was minimal
in Taxol-treated GFP-Cnn embryos (D) as compared with the mock control animals
(C). See also Movie 5 at
jcs.biologists.org/supplemental.
|
|
View larger version (45K):
[in a new window]
|
Fig. 8. Flare particles are restricted by the actin cage. Rhodamine-actin was
injected into GFP-Cnn embryos to view the dynamics of the centrosomes and the
actin cytoskeleton live. In metaphase (030), the flare particles, indicated by
yellow arrows, moved to the actin cage boundary and no further. At telophase,
when the centrosomes approach each other in close proximity (297), actin
assembled densely at the centrosomes (see arrows) on the sides opposite to the
nucleus. Following division, actin caps formed over the centrosomes (474) and
in interphase the actin cage bound the flares (594). See also Movie 6 at
jcs.biologist.org/supplemental.
|
|
View larger version (73K):
[in a new window]
|
Fig. 9. Flare activity is not wholly dependent on actin assembly. GFP-Cnn embryos
were injected with the actin destabilizing drug cytochalasin-D. In the absence
of the actin cytoskeleton, the nuclear and centrosome divisions were not
inhibited. The drug apparently does not affect flare activity. The centrosomes
flared under these conditions and appeared to transfer flare particles more
readily from one centrosome to another (see arrowheads in 132 and 249). The
arrows in the figures indicate the direction of movement of the flare
particles located closest to the tips of the arrows. movies available at
jcs.biologists.org/supplemental.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2002
