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Figure S1. In glutharaldehyde-fixed C. elegans embryos daughter centrioles are
first observed at pronuclear migration. a, DIC images of C. elegans embryos at
different stages during the first mitotic division. Shown are embryos in meiosis,
pronuclear appearance, pronuclear migration, pronuclear rotation and at metaphase.
The position of the female (left) and male (right) pronuclei (blue circles) is indicative
of specific developmental stages. It is know that the centriole pair contributed by the
sperm upon fertilization has duplicated prior to metaphase, but the process by which
daughter centrioles assemble until now was unclear. b, Observation by DIC of two
isolated embryos at a late stage of pronuclear migration. Developing embryos were
stopped at specific stages by laser-induced glutharaldehyde fixation. c, Lowmagnification electron micrograph of the local destruction of the embryo’s egg shell
(black arrow). d, Thin-section electron microscopy of an embryo at pronuclear
appearance. Arrows mark the position of the centrioles. High-magnification views of
centrioles are shown in the right panels. e, Embryo at pronuclear migration, at this
stage small daughter centrioles (white arrowheads) are detectable. f, Daughter
centrioles are more defined at the end of pronuclear migration (white arrowheads). g,
Embryo during pronuclear rotation. h, Pair of centrioles at prometaphase. Scale bars,
10 µm (b), 2,5 µm (c), 10 µm (h, overview image), and 100 nm (h, highmagnification EM image).
Figure S2. Mating assays can be used to monitor the recruitment of centriole
proteins to the site of daughter centriole assembly. Schematic representation of the
mating assay used throughout this study. Wild-type males are mated to feminized
hermaphrodites expressing individual GFP-tagged centriole proteins. Antibodies to
SAS-4 are used to detect the centriole pair contributed by the sperm upon fertilization
and GFP or ZYG-1 antibodies used to monitor the recruitment of the maternal
proteins to the site of centriole assembly.
Figure S3. ZYG-1::GFP is recruited during meiosis.
a, Sperm from ZYG-1::GFP hermaphrodites were stained for DNA (blue), SAS-4
(red) and anti-GFP antibodies. Note that ZYG-1::GFP is not expressed in sperm. Bar
is 1 µm. b, Wild-type males were mated to feminized hermaphrodites expressing
ZYG-1::GFP. Antibodies to SAS-4 are used to detect the centriole pair contributed
by the sperm upon fertilization and GFP antibodies used to monitor the recruitment of
maternal ZYG-1::GFP.
DNA (Blue), microtubules (Green), SAS-4 and GFP
labellings are shown. Note that ZYG-1::GFP is first recruited to centriole during
meiosis and remains associated with centrioles through the first cell division. Shown
are embryos in meiosis, at pronuclear meeting at metaphase. Bar is 10 µm.
Figure S4. Electron tomography of staged one-cell embryos.
Preparation of staged one-cell embryo for high-pressure freezing/freeze substitution
and electron tomography. Fertilized embryos are first collected in capillary tubes and
their development monitored by light microscopy before transferring them at the
desired stage to a specimen holder for high-pressure freezing.
After freeze-
substitution the samples are thin-layer embedded in preparation for serial sectioning
and electron tomography.
Figure S5.
Quantification of 3D centriole models. a, Spatial distribution and
relative positioning of centriolar microtubules on the central tube of mother (upper
panel) and daughter centrioles (lower panel) at pronuclear rotation. The lengths of the
mother (red bar) and daughter (yellow bar) central tubes (CT) are indicated. The
lateral position of individual singlet microtubules (green bars) relative to the proximal
and distal ends of the tube are shown. A slight bias for assembly at the distal end of
daughter centrioles is observed. b, Quantification of centriole size during the first
mitotic division of C. elegans embryos. Central tube lengths and widths of mother
(left panel) and daughter (right panel) central tubes at pronuclear appearance (PNA),
pronuclear migration (PNM), during pronuclear rotation (PNR) and after rotation
(mitotis). Values are in nm +/- standard deviation. One tail t-tests assuming unequal
variance were performed on the wild-type datasets to determine if the differences in
length and width of mother and daughter centrioles at different stages of the first
division are significant.
The slight increase in mother centriole width observed
between PNA, PNM, PNR and Mitotic embryos is not statistically significant
(p>0.05). However, the increase in length of mother centrioles, and the increase in
length and width of daughter centrioles are statistically significant (p<0.05).
Figure S6. The use of the slicer tool to display centriole structure. Because a
tomographic slice is a sample through a 3-D volume, its orientation can be rotated to
obtain the best view of a particular organelle. To facilitate this process, the modeling
and display program 3dmod includes an image slicer window that displays a slice
extracted from the 3-D volume in any position or orientation. With this slicer tool,
analysis of the morphology of centriole components can be done by extracting a slice
of image data one voxel thick and adjusting its orientation to view the centriole in
cross or longitudinal view by adjusting the sliders about an x or y axis. Multiple
slicer windows can be displayed to show the position of the object in various
orientations (Figure S5, red cross). Using this approach, microtubules can be
unambiguously modeled (top) as well as the central tube of the centriole (bottom). In
addition, model contours can be drawn to define the width and lengths of centriole
components and their dimensions can be extracted using the IMODINFO program.
An example of centriole structures modeled in this way is shown in Supplemental
Slicer Movie 1 and 2.
Figure S7. Modelling centriole structures in 3dmod. a-b, The positions of the
centriole MTs were marked by extracting a slice of image data one voxel thick and
adjusting its orientation to contain the axis of the singlet microtubules in a single
view. Model contours were then drawn, marking the position of the 9 singlet
microtubules and central tube of the centriole (Figure S6 a,b; green and red lines,
respectively). c, A three-dimensional surface was then calculated from the model
contour data to create cylindrical meshes around the contour lines. These cylindrical
meshes were calculated using the IMODMESH program, using the pixel diameter of
the singlet microtubules or central tubes in the reconstructions. d, A threedimensional projection of the model showing the complete mother centriole (green
microtubules, red central tube) and the central tube of the daughter centriole at
pronuclear migration (yellow tube).
Supplementary Table 1.
Table 1. Quantitative analysis of centriole assembly in one-cell wild-type and RNAi
embryos.
a
Central tube; b Not all centrioles fully contained in the sections; One tail t-tests assuming
unequal variance were performed on the wild-type datasets to determine if the differences in length
and width of mother and daughter centrioles at different stages of the first division are significant.
The slight increase in mother centriole width observed between PNA, PNM, PNR and Mitotic
embryos is not statistically significant (p>0.05). However, the increase in length of mother centrioles,
and the increase in length and width of daughter centrioles are statistically significant (p<0.05).