Figure 4.
Figure 4. PAR domain maintenance does not require an intact actin cytoskeleton. (A and B) Treatment of permeable embryos with CD or latrunculin A leads to rapid disruption of the actomyosin cortex as visualized with NMY-2–GFP (A) or LifeAct–GFP (B) using spinning disk confocal microscopy of a cortical plane taken before and 2–3 min after drug treatment. (C) Treatment of permeable embryos expressing fluorescently tagged PAR-2 (green)/PAR-6 (red) with CD or latrunculin A does not lead to loss of PAR domains. Select wide-field images of the embryo midplane are shown (Videos 1 and 2). (D) PAR distributions several minutes after treatment with CD are similar to untreated embryos (compare with Fig. 2 A). Mean ± SD is shown (n = 6 anterior to posterior profiles). Similar measurements for latrunculin A are provided in Fig. S2. (E) The recovery of GFP–PAR-2 during FRAP is similar in embryos left untreated compared with embryos treated with either CD or latrunculin A. Box size was 4.1 × 4.1 µm. In each case, two to four FRAP curves were averaged and normalized to allow comparison (see Materials and methods). (F) Same as E, but for GFP–PAR-6 embryos with a 6.9 × 6.9–µm box size. (G) A kymograph of GFP–PAR-2 in a CD-treated embryo shows that the domain remains relatively stable until anaphase (Ana, dashed white line), when it undergoes a dramatic contraction. Time is relative to nuclear envelope breakdown. Distance is relative to the center of the PAR-2 domain. Select images show a PAR-2 domain before and after anaphase. The PAR-2 invaginations that accompany domain contraction are indicated (white arrow; Video 3). Black lines indicate the extent of the PAR-2 domain at the beginning of time series and are shown above and below the kymograph to facilitate size comparisons. (H) Same as G, but including nocodazole plus CD. Disruption of microtubules eliminates both PAR-2 invaginations and anaphase PAR-2 domain contraction. (I) After anaphase onset, the boundaries of both mCherry–PAR-2 (green) and GFP–PAR-6 (red) shift to the posterior in CD-treated embryos. Images of a CD-treated embryo before (Metaphase) and after anaphase onset (Anaphase) illustrate the posterior migration of both PAR-2 and PAR-6 domain boundaries as a result of invaginations. In the insets, identical 25 × 25–µm regions encompassing the boundary region are taken from images before and after anaphase as indicated, and channels are shown individually to demonstrate the shift of both domain boundaries. Bars: (A–C) 5 µm; (G–I) 10 µm.

PAR domain maintenance does not require an intact actin cytoskeleton. (A and B) Treatment of permeable embryos with CD or latrunculin A leads to rapid disruption of the actomyosin cortex as visualized with NMY-2–GFP (A) or LifeAct–GFP (B) using spinning disk confocal microscopy of a cortical plane taken before and 2–3 min after drug treatment. (C) Treatment of permeable embryos expressing fluorescently tagged PAR-2 (green)/PAR-6 (red) with CD or latrunculin A does not lead to loss of PAR domains. Select wide-field images of the embryo midplane are shown (Videos 1 and 2). (D) PAR distributions several minutes after treatment with CD are similar to untreated embryos (compare with Fig. 2 A). Mean ± SD is shown (n = 6 anterior to posterior profiles). Similar measurements for latrunculin A are provided in Fig. S2. (E) The recovery of GFP–PAR-2 during FRAP is similar in embryos left untreated compared with embryos treated with either CD or latrunculin A. Box size was 4.1 × 4.1 µm. In each case, two to four FRAP curves were averaged and normalized to allow comparison (see Materials and methods). (F) Same as E, but for GFP–PAR-6 embryos with a 6.9 × 6.9–µm box size. (G) A kymograph of GFP–PAR-2 in a CD-treated embryo shows that the domain remains relatively stable until anaphase (Ana, dashed white line), when it undergoes a dramatic contraction. Time is relative to nuclear envelope breakdown. Distance is relative to the center of the PAR-2 domain. Select images show a PAR-2 domain before and after anaphase. The PAR-2 invaginations that accompany domain contraction are indicated (white arrow; Video 3). Black lines indicate the extent of the PAR-2 domain at the beginning of time series and are shown above and below the kymograph to facilitate size comparisons. (H) Same as G, but including nocodazole plus CD. Disruption of microtubules eliminates both PAR-2 invaginations and anaphase PAR-2 domain contraction. (I) After anaphase onset, the boundaries of both mCherry–PAR-2 (green) and GFP–PAR-6 (red) shift to the posterior in CD-treated embryos. Images of a CD-treated embryo before (Metaphase) and after anaphase onset (Anaphase) illustrate the posterior migration of both PAR-2 and PAR-6 domain boundaries as a result of invaginations. In the insets, identical 25 × 25–µm regions encompassing the boundary region are taken from images before and after anaphase as indicated, and channels are shown individually to demonstrate the shift of both domain boundaries. Bars: (A–C) 5 µm; (G–I) 10 µm.

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