Supplementary Figure and Movie Legends
Supplementary Figure 1. Contractile polarity depends on the assembly of
pericentriolar material. A) Generating ruffle kymographs to asess the establishment of
contractile polarity. DIC images were recorded at 5 second intervals (step 1). The
apices of cortical ingressions were manually tracked (step 2). These ruffle positions
were projected onto a calculated ellipse (steps 3-5), approximating the embryo
periphery. Ruffles above and below the anterior-posterior axis were analyzed
separately. The elliptical ruffle positions were straightened to generate the x axis of the
graph (step 6). This procedure was performed at each time point (step 7). Lines
connect ruffles within nearest neighbor groups (step 8). B-F) Time-lapse images and
ruffle kymographs of contractile polarity in B) control, C) spd-5(RNAi), D) spd2(RNAi), E) -tubulin(RNAi), and F) -/-tubulin(RNAi) embryos (similar results were
obtained for nocodazole treatment and -tubulin(RNAi)/nocodazole; data not shown and
Supplementary Figure 4). Individual ruffles are shown by red lines. Embryo posterior
is to the right. Anterior (ANT) and posterior (POST), or the meiotic pole (MEIO) in
cases where polarity was not established, are indicated on the graphs. Bars = 10 µm. In
A,B,E, and F, times were standardized to the onset of posterior smoothing; in C and D,
times were assigned based on a similar cell cycle stage to control embryos at the time of
polarization, judged by the size of the male pronucleus and the time elapsed relative to
meiosis II.
Supplementary Figure 2. Centrosome-cortex juxtaposition occurs in spd-2(RNAi) and
spd-5(RNAi) embryos. Solid lines indicate the distance (µm) from the centrosome to
the nearest point on the cortex over time. The dotted horizontal line indicates the upper
limit of centrosome-cortex distances at the time of polarity initiation in control embryos
(4 m; see the text and Figure 1B). Depletion of -tubulin, SPD-2, or SPD-5 does not
prevent the centrosome from achieving cortical proximity, but polarity is not established
in spd-2(RNAi) and spd-5(RNAi) embryos (Figure 3CD; Supplementary Figure 1CD).
In -tubulin(RNAi) embryos, times were standardized to the onset of posterior
smoothing, indicating polarity establishment. In spd-5(RNAi) and spd-2(RNAi)
embryos, times were assigned based on a similar cell cycle stage to control embryos at
the time of polarization, judged by the size of the male pronucleus and the time elapsed
relative to meiosis II. GFP::PAR-2; GFP::SPD-2 worms were used for the analysis of
-tubulin(RNAi) and spd-5(RNAi); GFP::SAS-4 worms were used to analyze spd2(RNAi) effects. Only a subset of experiments is shown.
Supplementary Figure 3. Microtubules are separable from polarity establishment.
Depletion of -tubulin or SPD-2 results in reduced amounts of centrosomal
microtubules (Figure 4A), but differring effects on polarity establishment. GFP::tubulin embryos were recorded prior to and during the time frame of polarity initiation.
Centrosomal microtubules, when visible, are indicated by white arrows. Ruffles (white
asterisks) are indicated in the last panel in each time lapse series to document contractile
polarity establishment in control and -tubulin(RNAi) embryos and the absence of
contractile polarity in spd-2(RNAi) embryos. In control and -tubulin(RNAi) embryos,
times (minutes:seconds) were standardized to the onset of posterior smoothing,
indicating polarity establishment. In spd-2(RNAi) embryos, times were assigned based
on a similar cell cycle stage to control embryos at the time of polarization, judged by
the size of the male pronucleus and the time elapsed relative to meiosis II. Embryo
posterior is to the right. Bars = 10 µm.
Supplementary Figure 4. GFP::PAR-2 polarity and contractile polarity are established
without microtubules. Time-lapse GFP::PAR-2 images and kymographs of PAR-2
polarity (upper panel) and time-lapse DIC images and ruffle kymographs of contractile
polarity (lower panel) in -tubulin(RNAi) + 15 µM nocodazole-treated embryos. Solid
green lines: PAR-2 boundaries; dotted green lines: cortical PAR-2; blue dots: pronuclei.
Red lines indicate individual ruffles. Embryo posterior is to the right. Anterior (ANT)
and posterior (POST) are indicated on the graphs. Bars = 10 µm. Times were
standardized to the onset of posterior smoothing. Worms were preincubated in 15 µM
nocodazole in 1X M9 for ~ 1 hour prior to dissecting embryos into 15 µM nocodazole
in 0.1 M NaCl 4% sucrose.
Supplementary Table 1
Primers used for dsRNA production.
Supplementary Movie 1
Centrosomes are adjacent to the cortex at the time of polarity initiation. Time lapse
movie of a GFP::PAR-2; GFP::SPD-2 embryo prior to and during polarity
establishment. The movie plays at roughly 120X actual speed. Embryo posterior is to
the right.
Supplementary Movie 2
Centrosome ablation prevents polarity establishment. The GFP::SPD-2-labelled
centrosome (indicated by the red circle) was ablated (red "x"); GFP::PAR-2 and cortical
ruffling were used to assess polarity. The movie plays at roughly 120X actual speed.
The embryo meiotic pole is to the left.
Supplementary Movie 3
Centrosome ablation prevents polarity establishment. The GFP::SPD-2-labelled
centrosome (indicated by the red circle) was ablated (red "x"); GFP::PAR-2 was
monitored to assess polarity. A failure in polar body extrusion is evident following
centrosome ablation, although this defect was also observed occaisionally in control
ablation embryos. The movie plays at roughly 120X actual speed. The embryo meiotic
pole is to the left.
Supplementary Movie 4
Control ablation does not affect polarity establishment. An area of cytoplasm (indicated
by the red circle) in the vicinity of the GFP::SPD-2-labelled centrosome was irradiated
(red "x"), as for centrosome ablations. GFP::PAR-2 was used to assess polarity. The
centrosomal SPD-2 is photobleached by the ablation but returns several frames later.
The movie plays at roughly 120X actual speed. Embryo posterior is to the right.
Supplementary Movie 5
Centrosome ablation after polarity initiation does not affect the propagation of posterior
polarity. The GFP::SPD-2-labelled centrosomes (indicated by the red circle) were
ablated (red "x") during the expansion of the posterior domain (boundary indicated by
green arrows). GFP::PAR-2 was used to monitor polarity pre- and post-ablation. The
movie plays at roughly 120X actual speed. Embryo posterior is to the right.
Supplementary Movie 6
Centrosome ablation after polarity establishment does not affect the maintenance of
posterior polarity. The GFP::SPD-2-labelled centrosomes (indicated by the red circles)
were ablated (red "x") after the posterior domain had been formed (PAR-2 occupied
half the embryo cortex). GFP::PAR-2 was used to monitor polarity pre- and postablation. The movie plays at roughly 120X actual speed. Embryo posterior is to the
right.
Supplementary Movie 7
Cortical control ablation during polarity initiation does not affect the extension of
posterior polarity. A small region of the cortex (indicated by the red circle) was ablated
(red "x") during the formation of the posterior domain. GFP::PAR-2 was used to
monitor polarity pre- and post-ablation; GFP::SPD-2 marks the centrosomes. Cortical
damage, confirming the effectiveness of the ablation, can be seen later in the recording
(indicated by a green arrow) The movie plays at roughly 120X actual speed. Embryo
posterior is to the right.