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First published online 9 August 2006
doi: 10.1242/dev.02527

Development 133, 3507-3516 (2006)
Published by The Company of Biologists 2006
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CDC-42 and RHO-1 coordinate acto-myosin contractility and PAR protein localization during polarity establishment in C. elegans embryos

Stephanie Schonegg* and Anthony A. Hyman

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.


Figure 1
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  		Fig. 1. CDC-42 is required for the PAR-2 localization cycle. (A-C) Time-lapse images of GFP-PAR-2 polarity establishment in (A) control, (B) cdc-42(RNAi) and (C) rho-1(RNAi) embryos. Times (seconds) are relative to nuclear envelope breakdown. In this and subsequent figures, the embryos are approximately 50 µm in length; the embryo posterior is to the right. (A) In control embryos, GFP-PAR-2 localizes uniformly along the cortex around the time of meiosis (top). After meiosis, GFP-PAR-2 disappears from the cortex (middle) and becomes confined to the posterior pole (bottom). (B) In cdc-42(RNAi) embryos, GFP-PAR-2 localized uniformly at the cortex. (C) Rho-1(RNAi) did not affect the PAR-2 localization cycle.

 

Figure 2
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  		Fig. 2. Meiotic GFP-PAR-2 localization is independent of the centrosomal signal. Embryos expressing GFP-PAR-2 were stained for GFP, SPD-2, microtubules (MT, green) and DNA (blue). In spd-2(RNAi) embryos, GFP-PAR-2 did not localize to the cortex. However, in cdc-42(RNAi);spd-2(RNAi) embryos, GFP-PAR-2 was found uniformly on the cortex as observed for cdc-42(RNAi) alone.

 

Figure 3
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  		Fig. 3. CDC-42 and RHO-1 are required for PAR-6 localization. (A) Embryos expressing GFP-PAR-2 were stained for GFP, PAR-6, microtubules (MT, green) and DNA (blue). In control embryos, PAR-6 localizes to the anterior pole, whereas PAR-2 localizes to the posterior pole. In cdc-42(RNAi) embryos, PAR-6 is not detectable at the cortex and PAR-2 is found at the entire cortex. In cdc-42(RNAi);par-2(RNAi) embryos, PAR-6 relocalizes anteriorly, but only at reduced levels. (B) Time-lapse images of GFP-PAR-6 polarity establishment in control and rho-1(RNAi) embryos. Times (seconds) are relative to nuclear envelope breakdown.

 

Figure 4
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  		Fig. 4. Cortical YFP-CDC-42 dynamics in mutant and RNAi-depleted embryos. (A) Time-lapse images of cortical YFP-CDC-42 recordings after polarity establishment. Times (min.sec) are relative to polarity establishment, as assessed by the proximity of the male pronucleus to the cortex. (B) Kymographs of cortical YFP-CDC-42 time-lapse recordings for a period of 7-12 minutes. (C) Position of the edge of the cortical YFP-CDC-42 accumulation from the anterior pole 6 minutes after polarity establishment in control and par-3(it71) embryos.

 

Figure 5
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  		Fig. 5. Ruffle kymographs monitoring the establishment of contractile polarity over time. The position of cortical ruffles along the anterior (ANT)-posterior (POST) axis is projected onto a calculated ellipse. One half of the ellipse was straightened to generate the x-axis (see Materials and methods). (A) In control embryos, the cortex contracts uniformly after the completion of meiosis. During anteroposterior polarity establishment, the posterior cortex becomes cleared from contractions, whereas the anterior cortex continuous to ruffle. (B) Cdc-42(RNAi) did not prevent the establishment of the contractile polarity. Ruffles were deeper and persisted longer than in control embryos. (C-E) Rho-1(RNAi) (C), cdc-42(RNAi);rho-1(RNAi) (D) and ect-2(RNAi) (E) abolished contractile polarity establishment.

 

Figure 6
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  		Fig. 6. RHO-1 activity organizes NMY-2 into foci clusters. (A) Time-lapse images (surface view) of GFP-NMY-2 during polarity establishment of control, ect-2(RNAi and cdc-42(RNAi) embryos. Times (seconds) are relative to pronuclei appearance. (B) Images of the combined NMY-2-GFP;GFP-PAR-2 line (surface view) of a control (left) and an ect-2(RNAi) embryo (middle); cortical view of GFP-PAR-2 (right). GFP-PAR-2 labels the posterior cortex. (C) Tracking of GFP-foci in an ect-2(RNAi) embryo. The small foci moved concomitantly at the same time and with similar velocities (average velocity=0.17 µm/second).

 




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